Gadolinium chelate compounds for use in magnetic resonance imaging

ABSTRACT

A compound having the formula of tetragadolinium [4,10-bis(carboxylatomethyl)-7-{-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclo dodecan-1-yl]acetate wherein the stereochemistry at the chiral carbon of the four alanine substituents is selected from the group consisting of RRRR, SSSS, RSSS, RRSS, and RRRS stereoisomers, and racemic and diastereomeric mixtures of any thereof, or a tautomer, a hydrate, a solvate, or a salt thereof, or a mixture of same is described. The compounds may be used as an MRI contrast imaging agent.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation application of U.S. Ser. No.16/192,185, filed 15 Nov. 2018, which is a Continuation application ofU.S. Ser. No. 15/578,687, filed 30 Nov. 2017, now U.S. Pat. No.10,137,209, issued 27 Nov. 2018, which is a U.S. national stage filingunder 35 U.S.C. § 371 of International Application No.PCT/EP2016/062105, filed 30 May 2016, which claims priority to EuropeanPatent Application No. EP 15170658.7, filed 4 Jun. 2015, the disclosuresof each of which are incorporated in their entirety herein by thisreference.

FIELD OF THE INVENTION

The present invention relates to the items characterized in the patentclaims, namely to new high relaxivity extracellular gadolinium chelatesbased on low molecular weight core polyamines, to methods of preparingsaid compounds, to the use of said compounds as MRI contrast agents andto their use in a mammalian body.

BACKGROUND 1. Introduction

Nine gadolinium-based contrast agents (GBCAs) have been approved forclinical use: gadopentetate dimeglumine (Magnevist®), gadoteratemeglumine (Dotarem®), gadoteridol (ProHance®), gadodiamide (Omniscan®),gadobutrol (Gadovist®), gadoversetamide (OptiMARK®), gadoxetic acid(Primovist®), gadobenate dimeglumine (MultiHance®) and gadofosvesettrisodium (Vasovist®/Ablavar®). With the exception of gadoxetic acid,gadobenate dimeglumine and gadofosveset trisodium, the GBCAs exhibit astrictly extracellular passive distribution in the body and are excretedexclusively via the kidney.

Gadoxetic acid and gadobenate dimeglumine exhibit a differentpharmacokinetic profile than the other agents. In addition to theextracellular distribution, they are taken up and are also excretedpartially via the liver. This allows, besides the classical imagingpossibilities (e.g. central nervous system, angiography, extremities,heart, head/face/neck, abdomen and breast imaging), also liver imagingdue to the enhancement of liver parenchyma caused by the GBCAs uptake inhepatocytes.

In contrast to the other GBCAs gadofosveset trisodium shows no passivediffusion in the body and remains in the vascular space. The prolongedperiod in the blood vessels caused by the reversible binding to HSA(human serum albumin) allows high resolution MR angiographies.

The various GBCAs differ in their efficacy which is given by theirlongitudinal (r1) and transversal (r2) relaxivity and is dependent onmagnetic field strengths, temperature and different intrinsic factors ofthe metal chelates. The intrinsic relaxivity influencing parameters aremainly the number of water molecules directly bound to the gadolinium(so-called inner-sphere water, q), the mean residence time of the innersphere water molecules (τm), the number and residence times of watermolecules in the second hydration sphere (so-called second sphere water)and the rotational diffusion (τr) (Helm L. et. al., Future Med. Chem.2010; 2: 385-396). In terms of their relaxivity all the commerciallyavailable GBCAs are very similar to each other and derived from a rangeof 4 to 7 L mmol⁻¹s⁻¹.

Strategies for increasing the sensitivity of GBCAs are frequentlydescribed in the literature (Caravan P. et. al. Chem. Soc. Rev., 2006,35, 512-523, Helm et. al. Future Med. Chem. 2010; 2:385-396, Jacques V.Invest. Radiol. 2010; 45:613-624). One of the strategies is the increaseof the inner sphere water molecules (q) that are water molecules whichare directly coordinated to the gadolinium ion in the chelate. As theexamples of AAZTA and HOPO-based ligands show, the increase of the innersphere water molecules from one to two leads to a significant increasein relaxivity. Another strategy to increase the relaxivity is theslowing of the rotational diffusion of the molecule. The so-calledtumbling rate (τr, see introduction) describes the tumbling of themolecule in solution and is mainly affected by the molecular size andprotein binding of the GBCA (Merbach A. S. et. al., The Chemistry ofContrast Agents in Medical Magnetic Resonance Imaging, 2013, ISBN:978-1-119-99176-2).

A further important characteristic of the GBCAs is their complexstability. The potential of the GBCAs to release free toxic Gd³⁺ ions isa major safety issue and of utmost importance in particular for patientswith end-stage renal disease. Nephrogenic systemic fibrosis (NSF) is arare and serious syndrome that is associated with the exposure to GBCAsin patients with severe kidney failure. NSF involves fibrotic changes inthe skin and many organs. In 2010, the Food and Drug Administration(FDA) published revised labeling recommendations for four GBCAs whichhave been principally implicated in NSF, including gadodiamide(Omniscan®), gadobenate dimeglumine (MultiHance®), gadopentetatedimeglumine (Magnevist®) and gadoversetamide (OptiMARK®) (Yang L et. al.Radiology. 2012; 265:248-253). At first glance the stability of allGBCAs is very high, but significant differences exist between the linearand macrocyclic agents and between the ionic and nonionicrepresentatives of the linear agents. The macrocyclic GBCAs possess thehighest complex stabilities (Frenzel T. et. al. Invest. Radiol. 2008;43:817-828). Due to the better awareness of risk patients, the use oflower doses and more widespread use of the macrocyclic GBCAs theincidence of NSF has decreased in the last years (Wang Y. et. al.Radiology. 2011; 260:105-111 and Becker S. et. al. Nephron. Clin. Pract.2012; 121:c91-c94).

The crucial issue for clinical applications is in vivo stability. Thekinetic inertness combined with the thermodynamic stability isparticularly with regard to the risk of nephrogenic systemic fibrosis(NSF) the best predictor of the in vivo toxicity of q=2 chelates(Merbach A. S. et. al., The Chemistry of Contrast Agents in MedicalMagnetic Resonance Imaging, 2013, ISBN: 978-1-119-99176-2, page157-208). The complexes with q=2 show two-fold enhancement of relaxivitybut, unfortunately, they have a lower stability than q=1 compounds(Hermann P. et. al. Dalton Trans., 2008, 3027-3047).

2. Description of the Prior Art, Problem to be Solved and its Solution

Several macrocyclic compounds are described in the prior art.

EP1931673 B1 and EP2457914 B1 relate to pyDO3A (q=2), DO3A and DOTAcompounds comprising short aminoalcohol chains and metal complexes formedical imaging.Macrocyclic lanthanide DO3A- and DOTA-like GBCAs with high relaxivitiesare described in the prior art.Ranganathan R. S. et. al (Investigative Radiology 1998; 33:779-797)investigated the effect of multimerization on the relaxivity ofmacrocyclic gadolinium chelates. WO199531444 relates to monomeric andmultimeric compounds having enhanced relaxivities.U.S. Pat. No. 5,679,810 relates to linear oligomer polychelant compoundsand chelates formed therewith, having alternating chelant and linkermoieties bound together by amide or ester moieties, and to their use indiagnostic imaging.U.S. Pat. No. 5,650,133 relates to dichelants, in particular compoundshaving two macrocyclic chelant groups linked by a bridge containing anester or amide bond, and to metal chelates thereof, and to their use indiagnostic imaging.WO 97/32862 A1 describes gadolinium polychelants as magnetic resonanceimaging agents which are linking at least two units of chelant to theamino groups of a target carrier structure (like e.g. a protein,aminoacid or peptide).US 2007/202047 relates to gadolinium chelate compounds for use inmagnetic resonance imaging, which are derived from a chelating moleculeselected from 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid(DOTA) and diethylentriaminepentaacetic acid (DTPA), wherein at leastone of the carboxylic groups of the chelating molecule is reacted withan amine.GBCAs with higher relaxivity offer on the one hand the opportunity of asignificant dose reduction and on the other an increased sensitivity inthe MRI examination of many diseases using the standard dose (Giese) F.L. et. al. Eur. Radiol. 2010, 20:2461-2474).

However, there is an unmet medical need to provide GBCAs for general usein magnetic resonance imaging, which:

exhibit high relaxivity,

show a favorable pharmacokinetic profile,

are completely excreted,

are chemically stable,

exhibit high water solubility,

offer the potential for a significant dose reduction,

are suitable for imaging of different body regions, and

are very well-tolerated.

The state of the art described above does not describe the specific highrelaxivity extracellular gadolinium chelate compounds of general formula(I) of the present invention as defined herein, or a stereoisomer, atautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or amixture of same, as described and defined herein, and as hereinafterreferred to as “compounds of the present invention”.

It has now been found, and this constitutes the basis of the presentinvention, that said compounds of the present invention havesurprisingly and advantageous properties.

In particular, said compounds of the present invention have been foundto exhibit a balanced profile of a high relaxivity, a favorablepharmacokinetic profile, a complete excretion, a high stability, a highsolubility, the potential for a significant dose reduction and thepotential for whole body imaging, and they may therefore be used ascontrast agents for magnetic resonance imaging (MRI).

SUMMARY

The present invention describes a new class of high relaxivityextracellular gadolinium chelate complexes, methods for theirpreparation and their use as MRI contrast agents.

DESCRIPTION OF THE INVENTION

In accordance with a first aspect, the present invention coverscompounds of general formula (I), comprising 4, 5, 6, 7 or 8 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups,

in which:

represents a group selected from:

-   -   in which groups a and b represent, independently from each        other, an integer of 1 or 2; and,    -   in which groups * indicates the point of attachment of said        group with R¹;

-   R¹ represents, independently from each other, a hydrogen atom or a    group selected from:    -   R³,

in which groups * indicates the point of attachment of said group withA, with the proviso that only one of the substituents R¹ may represent ahydrogen atom;n represents an integer of 3 or 4;

-   R² represents, independently from each other, a hydrogen atom or a    methyl group;-   R³ represents a group selected from:

-   -   in which groups * indicates the point of attachment of said        group with the rest of the molecule;

-   R⁴ represents, independently from each other, a hydrogen atom or a    methyl group;

-   R⁵ represents, independently from each other, a hydrogen atom or a    methyl group;    or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or    a salt thereof, or a mixture of same.

The compounds of this invention may contain one or more asymmetriccenter, depending upon the location and nature of the varioussubstituents desired. Asymmetric carbon atoms may be present in the (R)or (S) configuration, which can result in racemic mixtures in the caseof a single asymmetric center, and in diastereomeric mixtures in thecase of multiple asymmetric centers. In certain instances, asymmetry mayalso be present due to restricted rotation about a given bond, forexample, the central bond adjoining two substituted aromatic rings ofthe specified compounds.

Preferred compounds are those which produce the more desirablebiological activity. Separated, pure or partially purified isomers andstereoisomers or racemic or diastereomeric mixtures of the compounds ofthis invention are also included within the scope of the presentinvention. The purification and the separation of such materials can beaccomplished by standard techniques known in the art.

The optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes, for example, by theformation of diastereoisomeric salts using an optically active acid orbase or formation of covalent diastereomers. Examples of appropriateacids are tartaric, diacetyltartaric, ditoluoyltartaric andcamphorsulfonic acid. Mixtures of diastereoisomers can be separated intotheir individual diastereomers on the basis of their physical and/orchemical differences by methods known in the art, for example, bychromatography or fractional crystallization. The optically active basesor acids are then liberated from the separated diastereomeric salts. Adifferent process for separation of optical isomers involves the use ofchiral chromatography (e.g., chiral HPLC columns), with or withoutconventional derivatization, optimally chosen to maximize the separationof the enantiomers. Suitable chiral HPLC columns are manufactured byDaicel, e.g., Chiracel OD and Chiracel OJ among many others, allroutinely selectable. Enzymatic separations, with or withoutderivatization, are also useful. The optically active compounds of thisinvention can likewise be obtained by chiral syntheses utilizingoptically active starting materials.

In order to limit different types of isomers from each other referenceis made to IUPAC Rules Section E (Pure Appl. Chem. 45, 11-30, 1976).

The present invention includes all possible stereoisomers of thecompounds of the present invention as single stereoisomers, or as anymixture of said stereoisomers, e.g. R- or S-isomers, or E- or Z-isomers,in any ratio. Isolation of a single stereoisomer, e.g. a singleenantiomer or a single diastereomer, of a compound of the presentinvention may be achieved by any suitable state of the art method, suchas chromatography, especially chiral chromatography, for example.

Further, the compounds of the present invention can exist as N-oxides,which are defined in that at least one nitrogen of the compounds of thepresent invention is oxidized. The present invention includes all suchpossible N-oxides.

-   -   The present invention also relates to useful forms of the        compounds as disclosed herein, such as metabolites, hydrates,        solvates, salts, in particular pharmaceutically acceptable        salts, and co-precipitates.

The compounds of the present invention can exist as a hydrate, or as asolvate, wherein the compounds of the present invention contain polarsolvents, in particular water, methanol or ethanol for example asstructural element of the crystal lattice of the compounds. The amountof polar solvents, in particular water, may exist in a stoichiometric ornon-stoichiometric ratio. In the case of stoichiometric solvates, e.g. ahydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc.solvates or hydrates, respectively, are possible. The present inventionincludes all such hydrates or solvates.

Further, the compounds of the present invention can exist in the form ofa salt. Said salt may be either an inorganic or organic addition salt,particularly any pharmaceutically acceptable inorganic or organicaddition salt, customarily used in pharmacy.

The term “pharmaceutically acceptable salt” refers to a relativelynon-toxic, inorganic or organic acid addition salt of a compound of thepresent invention. For example, see S. M. Berge, et al. “PharmaceuticalSalts,” J. Pharm. Sci. 1977, 66, 1-19. The production of especiallyneutral salts is described in U.S. Pat. No. 5,560,903.

Pharmaceutically acceptable salts of the compounds according to theinvention include salts of mineral acids and carboxylic acids, forexample, without being limited thereto, salts of hydrochloric acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, lacticacid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid,aspartic acid and glutamic acid.

Those skilled in the art will further recognize that acid addition saltsof the claimed compounds may be prepared by reaction of the compoundswith the appropriate inorganic or organic acid via any of a number ofknown methods.

The present invention includes all possible salts of the compounds ofthe present invention as single salts, or as any mixture of said salts,in any ratio.

In the present text, in particular in the Experimental Section, for thesynthesis of intermediates and of examples of the present invention,when a compound is mentioned as a salt form with the corresponding baseor acid, the exact stoichiometric composition of said salt form, asobtained by the respective preparation and/or purification process, is,in most cases, unknown.

This applies analogously to cases in which synthesis intermediates orexample compounds or salts thereof have been obtained, by thepreparation and/or purification processes described, as solvates, suchas hydrates with (if defined) unknown stoichiometric composition.

In accordance with a second embodiment of the first aspect, the presentinvention covers compounds of general formula (I), supra, comprising 4,5 or 6, gadolinium[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups, wherein:

-   represents a group selected from:

-   -   in which groups a and b represent, independently from each        other, an integer of 1 or 2; and,    -   in which groups * indicates the point of attachment of said        group with R¹;

-   R¹ represents, independently from each other, a hydrogen atom or a    group selected from:    -   R³

in which groups * indicates the point of attachment of said group withA, with the proviso that only one of the substituents R¹ may represent ahydrogen atom;

-   n represents an integer of 3 or 4;-   R² represents, independently from each other, a hydrogen atom or a    methyl group;-   R³ represents a group selected from:

-   -   in which groups * indicates the point of attachment of said        group with the rest of the molecule;

-   R⁴ represents, independently from each other, a hydrogen atom or a    methyl group;

-   R⁵ represents, independently from each other, a hydrogen atom or a    methyl group;

-   or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or    a salt thereof, or a mixture of same.

In accordance with a third embodiment of the first aspect, the presentinvention covers compounds of general formula (I), supra, comprising 4,5 or 6, gadolinium[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups, wherein:

-   represents a group selected from:

-   -   in which groups a and b represent an integer of 1; and,    -   in which groups * indicates the point of attachment of said        group with R¹

-   R¹ represents, independently from each other, a hydrogen atom or a    group selected from:    -   R³,

in which groups * indicates the point of attachment of said group withA, with the proviso that only one of the substituents R¹ may represent ahydrogen atom,

-   n represents an integer of 3 or 4,-   R² represents a hydrogen atom;-   R³ represents a group selected from:

-   -   in which groups * indicates the point of attachment of said        group with the rest of the molecule;

-   R⁴ represents a hydrogen atom;

-   R⁵ represents a hydrogen atom or a methyl group;

-   or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or    a salt thereof, or a mixture of same.

In accordance with a fourth embodiment of the first aspect, the presentinvention covers compounds of general formula (I), supra, comprising 4,5 or 6, gadolinium[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups, wherein:

-   represents a group selected from:

-   -   in which groups a and b represent an integer of 1; and,    -   in which groups * indicates the point of attachment of said        group with R¹;

-   R¹ represents, independently from each other, a hydrogen atom or a    group selected from:    -   R³

in which groups * indicates the point of attachment of said group withA, with the proviso that only one of the substituents R¹ may represent ahydrogen atom;

-   n represents an integer of 3 or 4;-   R² represents a hydrogen atom;-   R³ represents a group selected from:

-   -   in which groups * indicates the point of attachment of said        group with the rest of the molecule;

-   R⁴ represents a hydrogen atom;

-   R⁵ represents a methyl group;

-   or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or    a salt thereof, or a mixture of same.

In accordance with another aspect, the present invention coverscompounds of general formula (I),

in which:

-   represents a

group,

-   -   in which group * indicates the point of attachment of said group        with R¹;

-   R¹ represents a group R³;

-   n represents an integer of 4;

-   R² represents a hydrogen atom;

-   R³ represents a group selected from:

-   -   in which groups * indicates the point of attachment of said        group with the rest of the molecule;

-   R⁴ represents a hydrogen atom;

-   R⁵ represents a hydrogen atom or a methyl group;

-   or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or    a salt thereof, or a mixture of same.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 4, 5, 6, 7 or 8gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 4, 5 or 6 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 4 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 5 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 6 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 7 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), comprising 8 gadolinium[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]groups.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a group selected from:

-   -   in which groups a and b represent, independently from each        other, an integer of 1 or 2; and    -   in which groups * indicates the point of attachment of said        group with R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a group selected from:

-   -   in which groups a and b represent, independently from each        other, an integer of 1 or 2; and    -   in which groups * indicates the point of attachment of said        group with R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a group selected from:

in which groups a and b represent an integer of 1; and

in which groups * indicates the point of attachment of said group withR¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group,in which groups a and b represent, independently from each other, aninteger of 1 or 2; andin which group * indicates the point of attachment of said group withR¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group,in which groups a and b represent an integer of 1; and in which group *indicates the point of attachment of said group with R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group,in which group * indicates the point of attachment of said group withR¹, and R² represents a hydrogen atom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

represents a

group, in which group * indicates the point of attachment of said groupwith R¹.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other, a hydrogen atom or a group selected from:

R³,

in which groups * indicates the point of attachment of said group withA, with the proviso that only one of the substituents R¹ may represent ahydrogen atom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other a group selected from:

R³

in which groups * indicates the point of attachment of said group withA.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other a group selected from:

R³, and

in which groups * indicates the point of attachment of said group withA.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other a group selected from:

R³, and

in which group * indicates the point of attachment of said group with A.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other a group selected from:

R³, and

in which group * indicates the point of attachment of said group with A.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents a group R³.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R¹ represents a

group, in which group * indicates the point of attachment of said groupwith A.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R¹ represents a

group, in which group * indicates the point of attachment of said groupwith A.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R¹ represents a

group, in which group * indicates the point of attachment of said groupwith A.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other, a hydrogen atom or a R³ group, with theproviso that only one of the substituents R¹ may represent a hydrogenatom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R¹ represents, independently from each other, a hydrogen atom or a

group, in which group * indicates the point of attachment of said groupwith A, with the proviso that only one of the substituents R¹ mayrepresent a hydrogen atom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R¹ represents, independently from each other, a hydrogen atom or a

group, in which group * indicates the point of attachment of said groupwith A, with the proviso that only one of the substituents R¹ mayrepresent a hydrogen atom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R¹ represents,independently from each other, a hydrogen atom or a

group,in which group * indicates the point of attachment of said group with A,with the proviso that only one of the substituents R¹ may represent ahydrogen atom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: n represents an integer of3 or 4.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: n represents an integer of3.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: n represents an integer of4.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R² represents,independently from each other, a hydrogen atom or a methyl group.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R² represents a hydrogenatom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R² represents a methylgroup.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R³ represents a groupselected from:

in which groups * indicates the point of attachment of said group withthe rest of the molecule.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R³ represents a

group,in which group * indicates the point of attachment of said group withthe rest of the molecule.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R³ represents a

group;in which group * indicates the point of attachment of said group withthe rest of the molecule.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R³ represents a

group;in which group * indicates the point of attachment of said group withthe rest of the molecule;and R⁵ represents a hydrogen atom or a methyl group.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R³ represents a

group;in which group * indicates the point of attachment of said group withthe rest of the molecule;and R⁵ represents a hydrogen atom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein:

R³ represents a

group;in which group * indicates the point of attachment of said group withthe rest of the molecule;

and R⁵ represents a methyl group.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R⁴ represents,independently from each other, a hydrogen atom or a methyl group.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R⁴ represents hydrogenatom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R⁴ represents a methylgroup.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R⁵ represents,independently from each other, a hydrogen atom or a methyl group.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R⁵ represents hydrogenatom.

In a further embodiment of the above-mentioned aspect, the inventionrelates to compounds of formula (I), wherein: R⁵ represents a methylgroup.

It is to be understood that the present invention relates also to anycombination of the embodiments described above.

Another embodiment of the first aspect are compounds of formula (I)selected from the group consisting of:

-   Pentagadolinium    [4,10-bis(carboxylatomethyl)-7-{3,6,10,18,22,25-hexaoxo-26-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-14-[({2-[4,7,10-tris(carboxylato-methyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-9,19-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}-methyl)-4,7,11,14,17,21,24-heptaazaheptacosan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]-acetate,-   Hexagadolinium    [4,10-bis(carboxylatomethyl)-7-{3,6,10,15,19,22-hexaoxo-23-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,16-bis({[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}-methyl)-11-(2-{[3-{[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-propanoyl}amino)acetyl]amino}-2-({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraaza-cyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)propanoyl]amino}ethyl)-4,7,11,14,18,21-hexaazatetracosan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate,-   Tetragadolinium    [4,10-bis(carboxylatomethyl)-7-{3,6,12,15-tetraoxo-16-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}-methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate,-   Tetragadolinium    {4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}-methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate,-   Tetragadolinium    {4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}-methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate,-   Pentagadolinium    [4-(1-{[2-(bis{2-[({1,4-bis[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetra-azacyclododecan-1-yl]propanoyl}amino)acetyl]-1,4-diazepan-6-yl}carbonyl)amino]ethyl}-amino)-2-oxoethyl]amino}-1-oxopropan-2-yl)-7,10-bis(carboxylatomethyl)-1,4,7,10-tetra-azacyclododecan-1-yl]acetate,-   Hexagadolinium    2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,    2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-{ethane-1,2-diylcarbamoyl-1,4-diazepane-6,1,4-triyltris[(2-oxo-ethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}octadecaacetate,-   Hexagadolinium    2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,    2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-(1,4,7-triazonane-1,4,7-triyltris{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})    octadecaacetate,-   Tetragadolinium    2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-{1,4,7,10-tetraazacyclo-    dodecane-1,4,7,10-tetrayltetrakis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}dodecaacetate,-   Hexagadolinium    2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,    2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-{3,7,10-triazatricyclo[3.3.3.0^(1,5)]undecane-3,7,10-triyltris[carbonyl-(3,6,11,14-tetraoxo-4,7,10,13-tetraazahexadecane-8,2,15-triyl)di-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}octadecaacetate,-   Tetragadolinium    2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-(3,7,9-triazabicyclo-[3.3.1]nonane-3,7-diylbis{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})dodecaacetate,-   Tetragadolinium    {4,10-bis(carboxylatomethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxylato-methyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(carboxylato-methyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]propyl}amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate,    and-   Tetragadolinium    [4,10-bis(carboxylatomethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris(carboxylato-methyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate,    or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or    a salt thereof, or a mixture of same.

In accordance with another aspect, the present invention covers methodsof preparing compounds of the present invention, said methods comprisingthe steps as described in the Experimental Section herein.

In accordance with a further aspect, the present invention coversintermediate compounds which are useful for the preparation of thecompounds of general formula (I), supra.

Particularly, the invention covers compounds of general formula (II-a):

in which

is as defined for the compounds of general formula (I), supra, and n′represents an integer of 2, 3 and 4, and salts thereof; and compounds ofgeneral formula (II-b):

in which

is as defined for the compounds of general formula (I), supra, and n′represents an integer of 2, 3 and 4, and salts thereof; and compounds ofgeneral formula (II-c):

in which

is as defined for the compounds of general formula (I), supra, and n′represents an integer of 2, 3 and 4, and salts thereof.

More particularly still, the present invention covers the intermediatecompounds which are disclosed in the example section of this text,infra.

In accordance with a further aspect, the present invention covers theuse of the compounds of general formula (II-a):

in which

is as defined for the compounds of general formula (I), supra, and n′represents an integer of 2, 3 and 4, and salts thereof, for thepreparation of a compound of general formula (I) as defined supra.

In accordance with a further aspect, the present invention covers theuse of the compounds of general formula (II-b):

in which

is as defined for the compounds of general formula (I), supra, and n′represents an integer of 2, 3 and 4, and salts thereof, for thepreparation of a compound of general formula (I) as defined supra.

In accordance with a further aspect, the present invention covers theuse of the compounds of general formula (II-c):

in which

is as defined for the compounds of general formula (I), supra, and n′represents an integer of 2, 3 and 4, and salts thereof, for thepreparation of a compound of general formula (I) as defined supra.

In accordance with a further aspect, the present invention covers theuse of the compounds of general formula (III):

in which R⁵ is as defined for the compounds of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) infra, for the preparation of acompound of general formula (I) as defined supra.

In accordance with a further aspect, the present invention covers theuse of the compounds of general formula (IV):

in which R⁴ is as defined for the compounds of general formula (I),supra, and

represents a tetraamine as defined for the compounds of general formula(I), supra, for the preparation of a compound of general formula (I) asdefined supra.

Another aspect of the invention is the use of a compound of generalformula (I) for diagnostic imaging.

Preferably, the use of a compound of the invention in the diagnosis isperformed using magnetic resonance imaging (MRI).

Other aspects of the invention are compounds of general formula (I) foruse in diagnostic imaging.

Other aspects of the invention are compounds of general formula (I) foruse in magnetic resonance imaging (MRI).

The invention also contains compounds of general formula (I) for themanufacture of diagnostic agents.

Another aspect of the invention is the use of the compounds of generalformula (I) or mixtures thereof for the manufacture of diagnosticagents.

Another aspect of the invention is the use of the compounds of generalformula (I) or mixtures thereof for the manufacture of diagnostic agentsfor magnetic resonance imaging (MRI).

Another aspect of the invention is a method of imaging body tissue in apatient, comprising the steps of administering to the patient aneffective amount of one or more compounds of general formula (I) in apharmaceutically acceptable carrier, and subjecting the patient to NMRtomography. Such a method is described in U.S. Pat. No. 5,560,903.

For the manufacture of diagnostic agents, for example the administrationto human or animal subjects, the compounds of general formula (I) ormixtures will conveniently be formulated together with pharmaceuticalcarriers or excipient. The contrast media of the invention mayconveniently contain pharmaceutical formulation aids, for examplestabilizers, antioxidants, pH adjusting agents, flavors, and the like.Production of the diagnostic media according to the invention is alsoperformed in a way known in the art, see U.S. Pat. No. 5,560,903. Theymay be formulated for parenteral or enteral administration or for directadministration into body cavities. For example, parenteral formulationscontain a sterile solution or suspension in a dose of 0.0001-5 mmolgadolinium/kg body weight, especially 0.005-0.5 mmol gadolinium/kg bodyweight of the compound of formula (I) according to this invention. Thus,the media of the invention may be in conventional pharmaceuticalformulations such as solutions, suspensions, dispersions, syrups, etc.in physiologically acceptable carrier media, preferably in water forinjections. When the contrast medium is formulated for parenteraladministration, it will be preferably isotonic or hypertonic and closeto pH 7.4.

In a further aspect, the invention is directed to a method of diagnosingand health monitoring of patients. This method comprises a)administering to a human in need of such diagnosis a compound of theinvention for detecting the compound in the human as described above andherein, and b) measuring the signal arising from the administration ofthe compound to the human, preferably by magnetic resonance imaging(MRI).

General Synthesis

The compounds according to the invention can be prepared according tothe following schemes 1 through 12.

The schemes and procedures described below illustrate synthetic routesto the compounds of general formula (I) of the invention and are notintended to be limiting. It is obvious to the person skilled in the artthat the order of transformations as exemplified in the schemes can bemodified in various ways. The order of transformations exemplified inthe schemes is therefore not intended to be limiting. Appropriateprotecting groups and their introduction and cleavage are well-known tothe person skilled in the art (see for example T. W. Greene and P. G. M.Wuts in Protective Groups in Organic Synthesis, 3^(rd) edition, Wiley1999). Specific examples are described in the subsequent paragraphs.

The term “amine-protecting group” as employed herein by itself or aspart of another group is known or obvious to someone skilled in the art,which is chosen from but not limited to a class of protecting groupsnamely carbamates, amides, imides, N-alkyl amines, N-aryl amines,imines, enamines, boranes, N—P protecting groups, N-sulfenyl, N-sulfonyland N-silyl, and which is chosen from but not limited to those describedin the textbook Greene and Wuts, Protecting groups in Organic Synthesis,third edition, pages 494-653, included herewith by reference. The“amine-protecting group” is preferably carbobenzyloxy (Cbz),p-methoxybenzyl carbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC),9-fluorenylmethyloxycarbonyl (FMOC), benzyl (Bn), p-methoxybenzyl (PMB),3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), triphenylmethyl(Trityl), methoxyphenyl diphenylmethyl (MMT) or the protected aminogroup is a 1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl (phthalimido) or anazido group.

The term “carboxyl-protecting group” as employed herein by itself or aspart of another group is known or obvious to someone skilled in the art,which is chosen from but not limited to a class of protecting groupsnamely esters, amides and hydrazides, and which is chosen from but notlimited to those described in the textbook Greene and Wuts, Protectinggroups in

Organic Synthesis, third edition, pages 369-453, included herewith byreference. The “carboxyl-protecting group” is preferably methyl, ethyl,propyl, butyl, tert-butyl, allyl, benzyl, 4-methoxybenzyl or4-methoxyphenyl.

The contents of the documents which are cited herein are herebyincorporated by reference.

A route for the preparation of compounds of general formula (I-a) isdescribed in Scheme 1.

Scheme 1: Route for the preparation of compounds of general formula(I-a), wherein

and R⁵ have the meaning as given for general formula (I), supra, n′represents an integer of 2, 3 and 4, and PG represents anamine-protecting group, such as for example a tert-butyloxycarbonylgroup (BOC) or a group as defined below.

The starting materials 1 are either commercially available polyamines orsalts thereof [for example CAS 111-40-0, CAS 28634-67-5, CAS 4730-54-5,CAS 4742-00-1, CAS 294-90-6] or polyamines or salts thereof which areknown from the literature, or which can be prepared in analogy tocompounds which are described in the literature or in the experimentalpart, infra [for example CAS 41077-50-3].

A triamine or tetraamine 1 or a salt thereof is reacted with a protected3-amino-2-(aminomethyl)propionic acid 2-a, [for example CAS 496974-25-5]or a salt thereof, leading to an intermediate 3-a. Suitableamine-protecting groups for 3-amino-2-(aminomethyl)propionic acid arefor example carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz orMeOZ), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC),benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM),p-methoxyphenyl (PMP), triphenylmethyl (Trityl), methoxyphenyldiphenylmethyl (MMT) or the protected amino group is a1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl(phthalimido) or an azido group.The coupling reaction of polyamines 1 with propionic acid derivatives2-a is carried out employing standard peptide coupling conditions, suchas for example coupling in the presence of HATU andN,N-diisopropylethylamine, in a suitable solvent such as for exampleN,N-dimethylformamide, in a temperature range from room temperature upto 80° C., to furnish the intermediates of general formula 3-a.

Deprotection of intermediates of general formula 3-a leading tointermediates of general formula (II-a) or salts thereof is performed inanalogy to methods described in the textbook Greene and Wuts, Protectinggroups in Organic Synthesis, second edition, pages 309-405, includedherewith by reference. The amine-protecting group tert-butyloxycarbonyl(BOC) is removed by dissolving a BOC-protected intermediate of generalformula 3-a in a suitable solvent, such as for example an alcohol,tetrahydrofuran, dioxane or N,N-dimethylformamide, or a mixture thereof,by adding suitable acids, such as for example aqueous hydrochloric orhydrobromic acid or trifluoroacetic acid in organic solvents likedichloromethane. The deprotection reaction is carried out attemperatures ranging from room temperature to the boiling point of therespective solvent or solvent mixture, preferably the reaction iscarried out at temperatures ranging from room temperature to 80° C.

Intermediates of general formula (II-a) or salts thereof are reactedwith Gd-complexes of the general formula (III), which are activated by aleaving group (LG), such as for example pentafluorophenol,4-nitrophenol, 1-hydroxypyrrolidine-2,5-dione, hydroxybenzotriazole or3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, leading to compounds of thegeneral formula (I-a). The preparation of activated esters is well knownto the person skilled in the art and is described in detail for exampleby C.A. Montalbetti and V. Falque in Tetrahedron 61 (2005), 10827-10852.For example, the preparation of gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetateis described in detail in WO 2001/051095 A2. The reaction ofintermediates of general formula (II-a) with the activated Gd-complexesof general formula (III) is carried out in a suitable solvent, such asfor example dimethyl sulfoxide, N,N-dimethylformamide, pyridine or amixture thereof, optionally the reaction is carried out in the presenceof a base. Suitable bases are for example trialkylamines, such as forexample triethylamine or N,N-diisopropylethylamine. The reaction iscarried out at temperatures ranging from room temperature to 100° C.,preferably the reaction is carried out at temperatures ranging from 50°C. to 70° C.

A route for the preparation of compounds of general formula (I-b) isdescribed in Scheme 2.

Scheme 2: Route for the preparation of compounds of general formula(I-b), wherein

and R⁵ have the meaning as given for general formula (I), supra, n′represents an integer of 2, 3 and 4, and PG represents anamine-protecting group, such as for example a tert-butyloxycarbonylgroup (BOC) or a group as defined for the synthesis of the compounds ofthe general formula (I-a) supra.

The compounds of general formula (I-b) are synthesized in analogy to thecompounds of general formula (I-a), as described above.

The starting materials 1 are either commercially available polyamines orsalts thereof [for example CAS 111-40-0, CAS 28634-67-5, CAS 4730-54-5,CAS 4742-00-1, CAS 294-90-6] or polyamines or salts thereof which areknown from the literature, or which can be prepared in analogy tocompounds which are described in the literature or in the experimentalpart, infra [for example CAS 41077-50-3].

A triamine or tetraamine 1 or a salt thereof is reacted with a protected2,3-diaminopropionic acid 2-b [for example CAS 201472-68-6] or a saltthereof, to furnish an intermediate of general formula 3-b, which afterdeprotection furnishes an intermediate of general formula (II-b) or asalt thereof. In the final step an intermediate of general formula(II-b) or a salt thereof is reacted with a Gd-complex of the generalformula (III), leading to a compound of the general formula (I-b).

A route for the preparation of compounds of general formula (I-c) isdescribed in Scheme 3.

Scheme 3: Route for the preparation of compounds of general formula(I-c), wherein

and R⁵ have the meaning as given for general formula (I), supra, n′represents an integer of 2, 3 and 4, and PG represents anamine-protecting group, such as for example a tert-butyloxycarbonylgroup (BOC) or a group as defined for the synthesis of the compounds ofthe general formula (I-a) supra.

The compounds of general formula (I-c) are synthesized in analogy to thecompounds of general formula (I-a), as described above.

The starting materials 1 are either commercially available polyamines orsalts thereof [for example CAS 111-40-0, CAS 28634-67-5, CAS 4730-54-5,CAS 4742-00-1, CAS 294-90-6] or polyamines or salts thereof which areknown from the literature, or which can be prepared in analogy tocompounds which are described in the literature or in the experimentalpart, infra [for example CAS 41077-50-3].

A triamine or tetraamine 1 or a salt thereof is reacted with a protected1,4-diazepane-6-carboxylic acid 2-c, which can be synthesized asdescribed in the experimental part infra, starting from methyl1,4-dibenzyl-1,4-diazepane-6-carboxylate [see U.S. Pat. No. 5,866,562],to furnish an intermediate of general formula 3-c, which afterdeprotection furnishes an intermediate of general formula (II-c) or asalt thereof. In the final step an intermediate of general formula(II-c) or a salt thereof is reacted with a Gd-complex of the generalformula (III), leading to a compound of the general formula (I-c).

A route for the preparation of compounds of general formula (I-d) isdescribed in Scheme 4.

Scheme 4: Route for the preparation of compounds of general formula(I-d), wherein R⁵ has the meaning as given for general formula (I),supra,

represents a tetraamine as given for general formula (I), supra, and LGrepresents an activating leaving group, such as for example4-nitrophenol, or a group as defined for the synthesis of the compoundsof the general formula (I-a) supra.

The starting materials 4 are either commercially available tetraaminesor salts thereof [for example CAS 4742-00-1, CAS 294-90-6] ortetraamines or salts thereof which are known from the literature, orwhich can be prepared in analogy to compounds which are described in theliterature.

A tetraamine 4 or a salt thereof is reacted with a Gd-complex of thegeneral formula (III), which is activated by a leaving group (LG), suchas for example pentafluorophenol, 4-nitrophenol,1-hydroxypyrrolidine-2,5-dione, hydroxybenzotriazole or3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, leading to a compound of thegeneral formula (I-d). The preparation of activated esters is well knownto the person skilled in the art and is described in detail for exampleby C.A. Montalbetti and V. Falque in Tetrahedron 61 (2005), page10827-10852. For example, the preparation of gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclodode-cane-1,4,7-triyl]triacetateis described in detail in WO 2001/051095 A2. The reaction of polyamine 4or a salt thereof with the activated Gd-complexes of general formula(III) is carried out in a suitable solvent, such as for example dimethylsulfoxide, N,N-dimethylformamide, pyridine or a mixture thereof,optionally the reaction is carried out in the presence of a base.Suitable bases are for example trialkylamines, such as for exampletriethylamine or N,N-diisopropylethylamine. The reaction is carried outat temperatures ranging from room temperature to 100° C., preferably thereaction is carried out at temperatures ranging from 50° C. to 70° C.

A route for the preparation of compounds of general formula (I-e) isdescribed in Scheme 5.

Scheme 5: Route for the preparation of compounds of general formula(I-e), wherein R⁴ has the meaning as given for general formula (I),supra,

represents a tetraamine as given for general formula (I), supra, and LGrepresents an activating leaving group, such as for example4-nitrophenol, or a group as defined for the synthesis of the compoundsof the general formula (I-a) supra.

The starting materials 4 are either commercially available tetraaminesor salts thereof [for example CAS 4742-00-1, CAS 294-90-6] ortetraamines or salts thereof which are known from the literature, orwhich can be prepared in analogy to compounds which are described in theliterature.

A tetraamine 4 or a salt thereof is reacted with a[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivative 5, which is activated by a leaving group (LG), such asfor example pentafluorophenol, 4-nitrophenol,1-hydroxypyrrolidine-2,5-dione [for example, the synthesis oftri-tert-butyl2,2′,2″-(10-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetateis described in detail by Cong Li et al., J. Am. Chem. Soc. 2006, 128,p. 15072-15073; S3-5 and Galibert et al., Biorg. and Med. Chem. Letters20 (2010), 5422-5425] or hydroxybenzotriazole, leading to anintermediate 6. The preparation of activated esters is well known to theperson skilled in the art and is described in detail for example by C.A.Montalbetti and V. Falque in Tetrahedron 61 (2005), 10827-10852. Thecoupling reaction of polyamines 4 with[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivatives 5 is carried out in a suitable solvent, such as forexample N,N-dimethylformamide or dimethyl sulfoxide, or a mixturethereof, in a temperature range from room temperature up to 80° C., tofurnish the intermediates 6. Cleavage of the carboxyl-protecting groupsof intermediates 6 to yield the intermediates of general formula (IV)can be achieved as described in the textbook Greene and Wuts, Protectinggroups in Organic Synthesis, second edition, pages 245-247. Thedeprotection is, for example, performed by dissolving and stirring ofintermediates 6 in trifluoroacetic acid at room temperature for severalhours. The complexation of intermediates of general formula (IV) withsuitable gadolinium (III) compounds or salts, such as for examplegadolinium trioxide, gadolinium triacetate or hydrates of gadoliniumtriacetate, gadolinium trichloride or gadolinium trinitrate, is wellknown to a person skilled in the art. The intermediates of generalformula (IV) are dissolved in water and after adding of suitablegadolinium (III) compounds the resulting mixtures are stirred in atemperature range from room temperature up to 100° C. at pH=1-7 forseveral hours, to furnish the compounds of general formula (I-e).Intermediates of general formula (IV) are, for example, dissolved inwater, gadolinium triacetate tetrahydrate is added, the pH is adjustedto 3.5-5.5 by addition of a suitable base, such as for example aqueoussodium hydroxide solution. The reaction is carried out at temperaturesranging from 50° C. to 80° C., leading to compounds of general formula(I-e).

A route for the preparation of compounds of general formula (I-f) isdescribed in Scheme 6.

Scheme 6: Route for the preparation of compounds of general formula(I-f), wherein n′ represents an integer of 2, if

represents a triamine as defined supra, or n′ represents an integer of3, if

represents a tetraamine as defined supra, and R⁵ has the meaning asgiven for general formula (I), supra, and LG represents activatingleaving groups, such as for example 4-nitrophenol or a group as definedbelow.

Intermediates of general formula (II-a) or salts thereof, as describedin Scheme 1 and in the experimental part infra, wherein n′ represents aninteger of 2 and

represents a triamine core as defined supra, or intermediates of generalformula (II-a) or salts thereof, wherein n′ represents an integer of 3and

represents a tetraamine core as defined supra, are reacted withGd-complexes of the general formula (III), which are activated by aleaving group (LG), such as for example pentafluorophenol,4-nitrophenol, 1-hydroxypyrrolidine-2,5-dione, hdyroxybenzotriazole or3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, leading to compounds of thegeneral formula (I-f). The preparation of activated esters is well knownto the person skilled in the art and is described in detail for exampleby C.A. Montalbetti and V. Falque in Tetrahedron 61 (2005), 10827-10852.For example, the preparation of gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetateis described in detail in WO 2001/051095 A2. The reaction ofintermediates of general formula (II-a) or salts thereof with theactivated Gd-complexes of general formula (III) is carried out in asuitable solvent, such as for example dimethyl sulfoxide,N,N-dimethylformamide, pyridine or a mixture thereof, optionally thereaction is carried out in the presence of a base. Suitable bases arefor example trialkylamines, such as for example triethylamine orN,N-diisopropylethylamine. The reaction is carried out at temperaturesranging from room temperature to 100° C., preferably the reaction iscarried out at temperatures ranging from 50° C. to 70° C.

A route for the preparation of compounds of general formula (I-g) isdescribed in Scheme 7.

Scheme 7: Route for the preparation of compounds of general formula(I-g), wherein n′ represents an integer of 2, if

represents a triamine as defined supra, or n′ represents an integer of 3if

represents a tetraamine as defined supra, and R⁵ has the meaning asgiven for general formula (I), supra, and LG represents activatingleaving groups, such as for example 4-nitrophenol or a group as definedbelow.

The compounds of general formula (I-g) are synthesized in analogy to thecompounds of general formula (I-f), as described above.

Intermediates of general formula (II-b) or salts thereof, as describedin Scheme 2, wherein n′ represents an integer of 2 and

represents a triamine core as defined supra, or intermediates of generalformula (II-b) or salts thereof, wherein n′ represents an integer of 3and

represents a tetraamine core as defined supra, are reacted withGd-complexes of the general formula (III), which are activated by aleaving group (LG), such as for example pentafluorophenol,4-nitrophenol, 1-hydroxypyrrolidine-2,5-dione, hydroxybenzotriazole or3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, leading to compounds of thegeneral formula (I-g).

A route for the preparation of compounds of general formula (I-h) isdescribed in Scheme 8.

Scheme 8: Route for the preparation of compounds of general formula(I-h), wherein n′ represents an integer of 2, if

represents a triamine as defined supra, or n′ represents an integer of3, if

represents a tetraamine as defined supra, and R⁵ has the meaning asgiven for general formula (I), supra, and LG represents activatingleaving groups, such as for example 4-nitrophenol or a group as definedbelow.

The compounds of general formula (I-h) are synthesized in analogy to thecompounds of general formula (I-f), as described above.

Intermediates of general formula (II-c) or salts thereof, as describedin Scheme 3, wherein n′ represents an integer of 2 and

represents a triamine core as defined supra, or intermediates of generalformula (II-c) or salts thereof, wherein n′ represents an integer of 3and

represents a tetraamine core as defined supra, are reacted withGd-complexes of the general formula (III), which are activated by aleaving group (LG), such as for example pentafluorophenol,4-nitrophenol, 1-hydroxypyrrolidine-2,5-dione, hydroxybenzotriazole or3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, leading to compounds of thegeneral formula (I-h).

A route for the preparation of compounds of general formula (I-k) isdescribed in Scheme 9.

Scheme 9: Route for the preparation of compounds of general formula(I-k), wherein

and R⁴ have the meaning as given for general formula (I), supra, n′represents an integer of 2, 3 and 4, LG represents activating leavinggroups, such as for example 1-hydroxypyrrolidine-2,5-dione, or a groupas defined for the synthesis of the compounds of the general formula(I-a) supra, and PG represents a carboxyl-protecting group, such as forexample a methyl or ethyl group.

The starting materials 1 are either commercially available polyamines orsalts thereof [for example CAS 111-40-0, CAS 28634-67-5, CAS 4730-54-5,CAS 4742-00-1, CAS 294-90-6] or polyamines or salts thereof which areknown from the literature, or which can be prepared in analogy tocompounds which are described in the literature or in the experimentalpart, infra [for example CAS 41077-50-3].

Diamines 7 or salts thereof are commercially available [for example CAS1417898-94-2] or can be synthesized by methods which are well known to aperson skilled in the art. Diamines 7 or salts thereof can be reactedwith a[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivative 5, which is activated by a leaving group (LG), such asfor example pentafluorophenol, 4-nitrophenol,1-hydroxypyrrolidine-2,5-dione [for example, the synthesis oftri-tert-butyl2,2′,2″-(10-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetateis described in detail by Cong Li et al., J. Am. Chem. Soc. 2006, 128,p. 15072-15073; S3-5 and. Galibert et al., Biorg. and Med. Chem. Letters2010, 20, p. 5422-5425] or hydroxybenzotriazole, leading tointermediates 8. The preparation of activated esters is well known tothe person skilled in the art and is described in detail for example byC.A. Montalbetti and V. Falque in Tetrahedron 2005, 61, 10827-10852. Theprotection group PG of intermediates 8 can be cleaved under basicconditions, such as for example by treatment with alkali metalhydroxides, such as for example lithium hydroxide, in water or a mixtureof water and tetrahydrofuran, to yield the corresponding salt of thecarboxylic acid. This salt can be coupled with polyamines 1 employingstandard peptide coupling conditions, such as for example coupling inthe presence of HATU and 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol in thepresence of N,N-diisopropylethylamine, in a suitable solvent, such asfor example dichloromethane, at room temperature, to furnish theintermediates of general formula (V). Cleavage of thecarboxyl-protecting groups of intermediates of general formula (V) canbe achieved employing standard conditions, such as for example, bydissolving and stirring of intermediates (V) in aqueous hydrochloricacid at room temperature. The subsequent complexation with suitablegadolinium (III) compounds or salts, such as for example gadoliniumtrioxide, gadolinium triacetate or hydrates of gadolinium triacetate,gadolinium trichloride or gadolinium trinitrate, is well known to aperson skilled in the art, and can, for example, be achieved by thereaction with suitable gadolinium (III) compounds in a temperature rangefrom room temperature up to 100° C. at pH=1-7 for several hours, tofurnish the compounds of general formula (I-k). The raw carboxylic acidsderived from the compounds of general formula (V) are, for example,reacted with gadolinium trioxide at 80° C., leading to compounds ofgeneral formula (I-k).

A route for the preparation of compounds of general formulae (I-m) and(I-n) is described in Scheme 10.

Scheme 10: Route for the preparation of compounds of general formulae(I-m) and (I-n), wherein

and R⁴ have the meaning as given for general formula (I), supra, n′represents an integer of 2, 3 and 4, LG represents activating leavinggroups, such as for example 1-hydroxypyrrolidine-2,5-dione, or a groupas defined for the synthesis of the compounds of the general formula(I-a) supra, and PG represents a carboxyl-protecting group, such as forexample a methyl or ethyl group.

When instead of the diamines of formula 7, as described in Scheme 9,diamines of formulae 9 and 10 or salts thereof are used in the analogoussynthesis as described in Scheme 9, the compounds of general formulae(I-m) and (I-n) can be obtained.

Diamines 9 or salts thereof are commercially available [for example CAS159029-33-1, CAS 440644-06-4] or can be synthesized by methods which arewell known to a person skilled in the art.

Diamines 10 or salts thereof are commercially available [for example CAS20610-20-2, CAS 6059-44-5] or can be synthesized by methods which arewell known to a person skilled in the art.

An alternative route to the one described in Scheme 4 for thepreparation of compounds of general formula (I-d) is described in Scheme11.

Scheme 11: Alternative route for the preparation of compounds of generalformula (I-d), wherein R⁵ has the meaning as given for general formula(I), supra,

represents a tetraamine as given for general formula (I), supra, and LGrepresents an activating leaving group, such as for example3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, or a group as defined for thesynthesis of the compounds of the general formula (I-a) supra.

The starting materials 4 are either commercially available tetraaminesor salts thereof [for example CAS 4742-00-1, CAS 294-90-6] ortetraamines or salts thereof which are known from the literature, orwhich can be prepared in analogy to compounds which are described in theliterature. The starting materials 14 are either commercially availableor known from the literature or can be synthesized in analogy tocompounds which are described in the literature, e.g. by step-wisealkylation of the cyclen core.

A tetraamine 4 or a salt thereof is reacted with an amino acidderivative 11, which is activated by a leaving group (LG), such as forexample 1-hydroxypyrrolidine-2,5-dione, pentafluorophenol, 4-nitrophenolor 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, leading to an intermediate 12.The preparation of activated esters is well known to the person skilledin the art and is described in detail for example by C.A. Montalbettiand V. Falque in Tetrahedron 61 (2005), 10827-10852. The couplingreactions of polyamines 4 with amino acid derivatives 11 are carried outin a suitable solvent, such as for example dichloromethane orN,N-dimethylformamide, in a temperature range from room temperature upto 50° C., to furnish the intermediates 12. Cleavage of the aminoprotecting groups (PG) of intermediates 12 to yield the intermediates 13can be achieved as described in the textbook Greene and Wuts, Protectinggroups in Organic Synthesis, second edition. In case oftert-butoxycarbonyl protecting groups the deprotection is, for example,performed by reacting intermediates 12 with HCl in CPME in a suitablesolvent, such as for example CPME or 1,4-dioxane or a mixture thereof ina temperature range from 0′C to room temperature for several hours.

A tetraamine 13 or a salt thereof is reacted with a[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivative 14, which is activated by a leaving group (LG), such asfor example 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, 4-nitrophenol or1-hydroxypyrrolidine-2,5-dione leading to an intermediate 15. Thecoupling reaction of tetraamines 13 with[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivatives 14 is carried out in a suitable solvent, such as forexample N,N-dimethylacetamide or dimethyl sulfoxide, or a mixturethereof, in a temperature range from room temperature to 80° C., tofurnish the intermediates 15.

Cleavage of the carboxyl-protecting groups of intermediates 15 to yieldthe intermediates of general formula (VI) can be achieved as describedin the textbook Greene and Wuts, Protecting groups in Organic Synthesis,second edition, pages 245-247. The deprotection is, for example,performed by dissolving and stirring of intermediates 15 intrifluoroacetic acid at room temperature for several hours.

The complexation of intermediates of general formula (VI) with suitablegadolinium (III) compounds or salts, such as for example gadoliniumtrioxide, gadolinium triacetate or hydrates of gadolinium triacetate,gadolinium trichloride or gadolinium trinitrate, is well known to aperson skilled in the art. The intermediates of general formula (VI) aredissolved in water and after adding of suitable gadolinium (III)compounds the resulting mixtures are stirred in a temperature range fromroom temperature up to 100° C. at pH=1-7 for several hours, to furnishthe compounds of general formula (I-d). Intermediates of general formula(VI) are, for example, dissolved in water, gadolinium triacetatetetrahydrate is added and the pH is adjusted to 3.5-5.5 by addition of asuitable base, such as for example aqueous sodium hydroxide solution.The reaction is carried out at temperatures ranging from 50° C. to 80°C., leading to compounds of general formula (I-d).

An alternative route to the one described in Scheme 4 for thepreparation of compounds of general formula (I-d) is described in Scheme12.

Scheme 12: Alternative route for the preparation of compounds of generalformula (I-d), wherein R⁵ has the meaning as given for general formula(I), supra,

represents a tetraamine as given for general formula (I), supra, and LGrepresents an activating leaving group, such as for example3H-[1,2,3]triazolo[4,5-b]pyridine-3-ol, or a group as defined for thesynthesis of the compounds of the general formula (I-a) supra.

The starting materials 4 are either commercially available tetraaminesor salts thereof [for example CAS 4742-00-1, CAS 294-90-6] ortetraamines or salts thereof which are known from the literature, orwhich can be prepared in analogy to compounds which are described in theliterature. The starting materials 16 are either known from theliterature or can be synthesized in analogy to compounds which aredescribed in the literature, e.g. by step-wise alkylation of the cyclencore.

A tetraamine 4 or a salt thereof is reacted with a[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivative 16, which is activated by a leaving group (LG), such asfor example 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol, 4-nitrophenol or1-hydroxypyrrolidine-2,5-dione leading to an intermediate 15. Thecoupling reaction of tetraamines 4 with[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid derivatives 16 is carried out in a suitable solvent, such as forexample N,N-dimethylformamide, to furnish the intermediates 16.

The complexation of intermediates of general formula (VI) with suitablegadolinium (III) compounds or salts, such as for example gadoliniumtrioxide, gadolinium triacetate or hydrates of gadolinium triacetate,gadolinium trichloride or gadolinium trinitrate, is well known to aperson skilled in the art. The intermediates of general formula (VI) aredissolved in water and after adding of suitable gadolinium (III)compounds the resulting mixtures are stirred in a temperature range fromroom temperature up to 100° C. at pH=1-7 for several hours, to furnishthe compounds of general formula (I-d). Intermediates of general formula(VI) are, for example, dissolved in water, gadolinium triacetatetetrahydrate is added and the pH is adjusted to 3.5-5.5 by addition of asuitable base, such as for example aqueous sodium hydroxide solution.The reaction is carried out at temperatures ranging from 50° C. to 80°C., leading to compounds of general formula (I-d).

In accordance with an embodiment, the present invention also relates toa method of preparing a compound of general formula (I-a) as definedsupra, said method comprising the step of allowing an intermediatecompound of general formula (II-a):

in which

is as defined for the compound of general formula (I), supra, and n′represents an integer of 2, 3 and 4, or a salt thereof, to react with acompound of general formula (III):

in which R⁵ is as defined for the compound of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) supra, thereby giving a compoundof general formula (I a):

in which

and R⁵ are as defined for the compound of general formula (I) supra, andn′ represents an integer of 2, 3 and 4.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-b) asdefined supra, said method comprising the step of allowing anintermediate compound of general formula (II-b):

in which

is as defined for the compound of general formula (I), supra, and n′represents an integer of 2, 3 and 4, or a salt thereof, to react with acompound of general formula (III):

in which R⁵ is as defined for the compound of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) supra, thereby giving a compoundof general formula (I-b):

in which

and R⁵ are as defined for the compound of general formula (I) supra, andn′ represents an integer of 2, 3 and 4.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-c) asdefined supra, said method comprising the step of allowing anintermediate compound of general formula (II-c):

in which

is as defined for the compound of general formula (I), supra, and n′represents an integer of 2, 3 and 4, or a salt thereof, to react with acompound of general formula (III):

in which R⁵ is as defined for the compound of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) supra, thereby giving a compoundof general formula (I-c):

in which

and R⁵ are as defined for the compound of general formula (I) supra, andn′ represents an integer of 2, 3 and 4.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-d) asdefined supra, said method comprising the step of allowing a compound offormula 4,

in which

is a tetraamine as defined for the compound of general formula (I),supra, or a salt thereof, to react with a compound of general formula(III):

in which R⁵ is as defined for the compound of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) supra, thereby giving a compoundof general formula (I-d):

in which R⁵ is as defined for the compound of general formula (I) supra,and

is a tetraamine as defined for the compound of general formula (I),supra.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-e) asdefined supra, said method comprising the step of allowing anintermediate compound of general formula (IV):

in which R⁴ is as defined for the compound of general formula (I),supra, and

is a tetraamine as defined for the compound of general formula (I),supra, to react with a gadolinium (III) compound, such as for examplegadolinium trioxide, gadolinium triacetate or hydrates of gadoliniumtriacetate, gadolinium trichloride or gadolinium trinitrate, or with asalt thereof, thereby giving a compound of general formula (I-e):

in which R⁴ is as defined for the compound of general formula (I),supra, and

is a tetraamine as defined for the compound of general formula (I),supra.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-f) asdefined supra, said method comprising the step of allowing anintermediate compound of general formula (II-a):

in which check numbers

is a triamine as defined for the compound of general formula (I), supra,and n′ represents an integer of 2, or a salt thereof, or in which

is a tetraamine as defined for the compound of general formula (I),supra, and n′ represents an integer of 3, or a salt thereof, to reactwith a compound of general formula (III):

in which R⁵ is as defined for the compound of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) supra, thereby giving a compoundof general formula (I-f):

in which R⁵ is as defined for the compound of general formula (I),supra, and in which

is a triamine as defined for the compound of general formula (I), supra,and n′ represents an integer of 2, or in which

is a tetraamine as defined for the compound of general formula (I),supra, and n′ represents an integer of 3.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-h) asdefined supra, said method comprising the step of allowing anintermediate compound of general formula (II-c):

in which

is a triamine as defined for the compound of general formula (I), supra,and n′ represents an integer of 2, or a salt thereof, or in which

is a tetraamine as defined for the compound of general formula (I),supra, and n′ represents an integer of 3, or a salt thereof, to reactwith a compound of general formula (III):

in which R⁵ is as defined for the compound of general formula (I),supra, and LG represents an activating leaving group, such as forexample 4-nitrophenol, or a group as defined for the synthesis of thecompounds of the general formula (I-a) supra, thereby giving a compoundof general formula (I-h):

in which R⁵ is as defined for the compound of general formula (I),supra, and in which

is a triamine as defined for the compound of general formula (I), supra,and n′ represents an integer of 2, or in which

is a tetraamine as defined for the compound of general formula (I),supra, and n′ represents an integer of 3.

In accordance with another embodiment, the present invention alsorelates to a method of preparing a compound of general formula (I-k) asdefined supra, said method comprising the step of allowing anintermediate compound of general formula (V):

in which

and R⁴ are as defined for the compound of general formula (I), supra,and n′ represents an integer of 2, 3 and 4, in a first step to reactwith an acid, such as for example aqueous hydrochloric acid, and in asecond step to react with a gadolinium (III) compound, such as forexample gadolinium trioxide, gadolinium triacetate or hydrates ofgadolinium triacetate, gadolinium trichloride or gadolinium trinitrate,or with a salt thereof, thereby giving a compound of general formula(I-k):

in which

and R⁴ are as defined for the compound of general formula (I), supra,and n′ represents an integer of 2, 3 and 4.

DESCRIPTION OF THE FIGURES

FIG. 1: shows the blood plasma kinetic of Example 3 versus Gadovist® inrats. The pharmacokinetic profile of Example 3 is comparable to that ofGadovist®.

FIG. 2: shows the evolution of the relative water proton paramagneticlongitudinal relaxation rate R₁ ^(p)(t)/R₁ ^(p)(0) versus time ofExample 3, Reference compound 1 (Gadovist®), Reference compound 2(Magnevist®) and Reference compound 3 (Primovist®). The stability ofExample 3 is comparable to the high stability macrocyclic Referencecompound 1 (Gadovist®).

FIGS. 3A to 3C: show the magnetic resonance angiography data in male NewZealand white rabbits: (FIG. 3A) 30 μmol Gd/kg bw Reference compound 1(Gadovist®); (FIG. 3B) 30 μmol Gd/kg bw Example 3 and (FIG. 3C) 100 μmolGd/kg bw Reference compound 1. The contrast enhancement of the low doseprotocol with Example 3 (FIG. 3B) is comparable to that of the standarddose of Reference compound 1 (FIG. 3C). Furthermore, the image qualityof the low dose protocol of Example 3 (FIG. 3B) is significantly betterthan the low dose protocol of Reference compound 1 (FIG. 3A). Theangiography study demonstrates the potential for Example 3 for asignificant dose reduction.

FIGS. 4A and 4B: MR images before and after administration of contrastagent. Representative images of the head and neck region before and 1.4min after administration of Example 3 (FIG. 4A) and reference compound 1(FIG. 4B). The strong signal enhancement is visible for example in theheart, the tongue and the neck muscle.

FIGS. 5A and 5B: MR images before and after administration of contrastagent. Representative images of the abdominal region before and 0.5 minafter administration of Example 3 (FIG. 5A) and reference compound 1(FIG. 5B). The strong signal enhancement is visible for example in theaorta, kidney, liver and spleen.

FIGS. 6A and 6B: MR images before and after administration of contrastagent. Representative images of the pelvis region before and 2.9 minafter administration of Example 3 (FIG. 6A) and reference compound 1(FIG. 6B). The strong signal enhancement is visible for example in thevascular system (vessels) and the extremity muscles.

FIG. 7: MRI signal enhancements for different body regions. Signalenhancement over time after administration of Example 3 and Referencecompound 1 (Gadovist®) for tongue, chops muscle, liver, spleen, aortaand extremity muscle. No differences in the time course of signalchanges were observed between Example 3 and reference compound 1. Thisdemonstrates identical pharmacokinetic properties and indicates thepotential of Example 3 for the imaging of different body regions. Asexpected from the approximately 2-fold higher relaxivity (see exampleA), the observed contrast enhancements of Example 3 were higher comparedto that of reference compound 1 (Gadovist®). The vertical bars representthe standard deviation.

FIGS. 8A and 8B: Correlation of tissue gadolinium concentration and MRIsignal enhancement. The gadolinium concentration was measured in tissuesamples of the brain, tongue, liver, spleen, blood and extremity muscle(muscle) and respective MRI signal changes determined in-vivo, afteradministration of Example 3 (FIG. 8A) and reference compound 1 (FIG.8B). The vertical and horizontal error bars represent the standarddeviation. The dotted lines represent the linear regression betweengadolinium concentration and MRI signal change.

FIGS. 9A and 9B: Diffusion of different contrast agents throughsemipermeable membranes (20 kDa). Dynamic CT measurements were performedto show the ability of different contrast agents to diffuse through asemipermeable membrane. (FIG. 9A and FIG. 9B) CT images of Example 1, 2,3, 4, 5 and 6 in comparison to that of Reference compound 1 (Gadovist®)and 4 (Gadomer). A representative measurement region for the signalevaluation over time is indicated in the image A1.

FIG. 10: Signal analysis of dynamic CT diffusion phantom study overtime. Signal in Hounsfield units (HU) over time of the dialysis cassettein fetal bovine solution for Example 1-6 and reference compounds 1 and 4demonstrate that contrary to Reference compound 4 (Gadomer), all of theinvestigated compound are able to pass the semipermeable membrane (20kDa).

FIGS. 11A and 11 B: Contrast-enhanced magnetic resonance images of GS9Lbrain tumors in rats (marked with white arrows). (FIG. 11A)Intraindividual comparison of Reference compound 1 (Gadovist®) andExample 3 at the same dose of 0.1 mmol Gd/kg body weight (bw). Example 3showed higher lesion-to-brain contrast and an excellent demarcation ofthe tumor rim. (FIG. 11B) Comparison of the Reference compound 1(Gadovist®) at 0.3 mmol Gd/kg bw and Example 3 at 0.1 mmol Gd/kw bw.Example 3 showed similar lesion-to-brain contrast at one third of thedose of Reference compound 1.

EXPERIMENTAL SECTION Abbreviations

ACN acetonitrile AUC area under the curve br broad signal (in NMR data)bw body weight CPME cyclopentyl methyl ether CPMGCarr-Purcell-Meiboom-Gill (MRI sequence) C_(Gd) concentration of thecompound normalized to the Gadolinium CI chemical ionization Cl_(tot)total clearance d day(s) DAD diode array detector DCM dichloromethaneDMF N,N-dimethylformamide DMSO dimethylsulfoxide DMSO-d₆ deuterateddimethylsulfoxide ECCM extracellular contrast media EI electronionization ELSD evaporative light scattering detector ESI electrosprayionization FBS fetal bovine serum h hour HATUN-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylidene]-N-methylmethanaminium hexafluorophosphate HCOOH formic acidHPLC high performance liquid chromatography HU Hounsfield units IRinversion recovery kDa kilo Dalton LCMS liquid chromatography-massspectroscopy ICP-MS Inductively coupled plasma mass spectrometry MRImagnetic resonance imaging MRT mean residence time MS mass spectrometrym multiplet min minute(s) NMR nuclear magnetic resonance spectroscopy:chemical shifts (δ) are given in ppm. r_(i) (where i = 1, 2)relaxivities in L mmol⁻¹ s⁻¹ Rt. retention time s singlet RC referencecompound R_(i) (where i = 1, 2) relaxation rates (1/T_(1,2)) R_(i(0))relaxation rate of the respective solvent T_(1,2) relaxation time TTesla t triplet t½ α plasma half-life, compartment V1 t½ β plasmahalf-life, compartment V2 t½ γ plasma half-life, compartment V3 TFAtrifluoroacetic acid THF tetrahydrofuran TI inversion time UPLC ultraperformance liquid chromatography V1 + V2 volume, compartments V_(c)(V1) volume, central compartment V1 V_(d,ss) volume of distribution atsteady state

Materials and Instrumentation

The chemicals used for the synthetic work were of reagent grade qualityand were used as obtained.

All reagents, for which the synthesis is not described in theexperimental section, are either commercially available, or are knowncompounds or may be formed from known compounds by known methods by aperson skilled in the art.

¹H-NMR spectra were measured in CDCl₃, D₂O or DMSO-d₆, respectively(room temperature, Bruker Avance 400 spectrometer, resonance frequency:400.20 MHz for ¹H or Bruker Avance 300 spectrometer, resonancefrequency: 300.13 MHz for ¹H. Chemical shifts are given in ppm relativeto sodium (trimethylsilyl)propionate-d₄ (D₂O) or tetramethylsilane(DMSO-d₆) as external standards (δ=0 ppm).

The compounds and intermediates produced according to the methods of theinvention may require purification. Purification of organic compounds iswell known to the person skilled in the art and there may be severalways of purifying the same compound. In some cases, no purification maybe necessary. In some cases, the compounds may be purified bycrystallization. In some cases, impurities may be stirred out using asuitable solvent. In some cases, the compounds may be purified bychromatography, particularly flash column chromatography, using forexample prepacked silica gel cartridges, e.g. Biotage SNAP cartridgesKP-Sil® or KP-NH® in combination with a Biotage autopurifier system(SP4° or Isolera Four®) and eluents such as gradients of hexane/ethylacetate or DCM/methanol. In some cases, the compounds may be purified bypreparative HPLC using for example a Waters autopurifier equipped with adiode array detector and/or on-line electrospray ionization massspectrometer in combination with a suitable prepacked reverse phasecolumn and eluents such as gradients of water and acetonitrile which maycontain additives such as trifluoroacetic acid, formic acid or aqueousammonia.

Examples were analysed and characterized by the following HPLC basedanalytical methods to determine characteristic retention time and massspectrum:

Method 1: UPLC (ACN-HCOOH):

Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEHC18 1.7 μm, 50×2.1 mm; eluent A: water+0.1% formic acid, eluent B:acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8mL/min; temperature: 60° C.; injection: 2 μl; DAD scan: 210-400 nm;ELSD.

Method 2: UPLC (ACN-HCOOH polar):

Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEHC18 1.7 μm, 50×2.1 mm; eluent A: water+0.1% formic acid, eluent B:acetonitrile; gradient: 0-1.7 min 1-45% B, 1.7-2.0 min 45-99% B; flow0.8 mL/min; temperature: 60° C.; injection: 2 μl; DAD scan: 210-400 nm;ELSD.

Method 3: UPLC (ACN-HCOOH Long Run):

Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEHC18 1.7 μm, 50×2.1 mm; eluent A: water+0.1% formic acid, eluent B:acetonitrile; gradient: 0-4.5 min 0-10% B; flow 0.8 mL/min; temperature:60° C.; injection: 2 μl; DAD scan: 210-400 nm; ELSD.

Method 4: UPLC (ACN-NH₃):

Instrument: Waters Acquity UPLC-MS ZQ2000; column: Acquity UPLC BEH C181.7 μm, 50×2.1 mm; Eluent A: water+0.2% ammonia, eluent B: acetonitrile;gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate 0.8 mL/min;temperature: 60° C.; injection: 1 μL; DAD scan: 210-400 nm; ELSD.

Method 5: LC-MS:

Instrument: Agilent 1290 UHPLCMS Tof; column: BEH C 18 (Waters) 1.7 μm,50×2.1 mm; eluent A: water+0.05 vol-% formic acid (99%), eluent B:acetonitrile+0.05% formic acid; gradient: 0-1.7 min 98-10% A, 1.7-2.0min 10% A, 2.0-2.5 min 10-98% A, flow 1.2 mL/min; temperature: 60° C.;DAD scan: 210-400 n m.

Example Compounds Example 1 Pentagadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,10,18,22,25-hexaoxo-26-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-14-[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-9,19-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-propanoyl}amino)acetyl]amino}methyl)-4,7,11,14,17,21,24-heptaazaheptacosan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

Example 1a Di-tert-butyl(2-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}propane-1,3-diyl)biscarbamate

3.60 g (11.3 mmol, 1 eq.)3-[(tert-butoxycarbonyl)amino]-2-{[(tert-butoxycarbonyl)amino]methyl}propanoicacid (see WO 2006/136460 A2) and 1.43 g (12.4 mmol, 1.1 eq.)1-hydroxypyrrolidine-2,5-dione were dissolved in 120 mL THF. To thereaction mixture was added dropwise a solution of 2.57 g (12.4 mmol, 1.1eq.) N,N-dicyclohexylcarbodiimide in 60 mL THF. After stirring for 3hours at room temperature, the resulting suspension was cooled to 0° C.and the precipitated urea was filtered off. The clear solution wasevaporated to dryness yielding 5.50 g (13.24 mmol, 117%) of the titlecompound.

UPLC (ACN-HCOOH): Rt.=1.15 min.

MS (ES⁺): m/z=416.3 (M+H)⁺.

Example 1 b Tert-butyl(7,17-bis{[(tert-butoxycarbonyl)amino]methyl}-2,2-dimethyl-4,8,16-trioxo-3-oxa-5,9,12,15-tetraazaoctadecan-18-yl)carbamate

4.70 g (11.3 mmol, 2.22 eq.) Di-tert-butyl(2-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}propane-1,3-diyl)biscarbamate (example 1 a) were dissolved in 120 mLTHF. To the reaction mixture was added dropwise a solution of 0.53 g(5.10 mmol, 1 eq.)N-(2-aminoethyl)ethane-1,2-diamine and 1.14 g (11.3mmol, 2.22 eq.) triethylamine in 40 mL THF. After stirring for 3 hoursat room temperature, the resulting suspension was diluted withdichloromethane. The organic solution was washed with aqueous sodiumhydroxide (0.1 M), with water, and was dried over sodium sulfate. Thecrude product was isolated by evaporation under reduced pressure and waspurified by silica gel chromatography yielding 2.81 g (3.99 mmol, 78%)of the title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ=1.36 (s, 36H), 2.39-2.47 (m, 3H), 2.52-2.58(m, 4H), 2.95-3.20 (m, 12H), 6.64 (t, 4H), 7.72 (t, 2H) ppm.

UPLC (ACN-HCOOH): Rt.=1.06 min.

MS (ES⁺): m/z=704.6 (M++H).

Example 1cN,N′-(Iminodiethane-2,1-diyl)bis[3-amino-2-(aminomethyl)propanamide]pentahydrochloride

600 mg (0.85 mmol) Tert-butyl(7,17-bis{[(tert-butoxycarbonyl)amino]methyl}-2,2-dimethyl-4,8,16-trioxo-3-oxa-5,9,12,15-tetraazaoctadecan-18-yl)carbamate(example 1b) were dissolved in 9.6 mL methanol and 2.85 mL aqueoushydrochloric acid (37%). The reaction mixture was heated under stirringfor 2 hours at 50° C. For isolation, the suspension was evaporated todryness yielding 423 mg (0.87 mmol, 102%) of the title compound.

¹H-NMR (400 MHz, D₂O): δ=3.04-3.15 (m, 2H), 3.17-3.27 (m, 8H), 3.29-3.38(m, 4H), 3.55 (t, 4H) ppm.

UPLC (ACN-HCOOH): Rt.=0.19 min.

MS (ES⁺): m/z=304.2 (M+H)⁺, free base.

Example 1 Pentagadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,10,18,22,25-hexaoxo-26-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-14-[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-9,19-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-propanoyl}amino)acetyl]amino}methyl)-4,7,11,14,17,21,24-heptaazaheptacosan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

150 mg (309 μmol, 1 eq.)N,N′-(Iminodiethane-2,1-diyl)bis[3-amino-2-(aminomethyl)-propanamide]pentahydrochloride(example 1c) were dissolved in 60 mL DMSO. After adding of 499 mg (3.86mmol, 12.5 eq.) N,N-diisopropylethylamine and 4.06 g (5.40 mmol, 17.5eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 15-20 mL. The concentrate waspoured in 400 mL ethyl acetate under stirring, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane, and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 668 mg (64%, 199 μmol) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.46 min.

MS (ES⁻): m/z (z=2)=1680.5 (M−2H)²⁻; (ES⁺): m/z (z=3)=1121.3 (M+H)³⁺,m/z (z=4)=841.4 [(M+H)⁴⁺.

Example 2

Hexagadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,10,15,19,22-hexaoxo-23-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,16-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-amino}methyl)-11-(2-{[3-{[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl]propanoyl}amino)acetyl]amino}-2-({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)propanoyl]-amino}ethyl)-4,7,11,14,18,21-hexaazatetracosan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

Example 2a Tert-butyl(12-{2-[(3-[(tert-butoxycarbonyl)amino]-2-{[(tert-butoxycarbonyl)amino]-methyl}propanoyl)amino]ethyl}-7,14-bis{[(tert-butoxycarbonyl)amino]methyl}-2,2-dimethyl-4,8,13-trioxo-3-oxa-5,9,12-triazapentadecane-15-yl)carbamate

890 mg (2.80 mmol, 3 eq.)3-[(Tert-butoxycarbonyl)amino]-2-{[(tert-butoxycarbonyl)amino]-methyl}propanoicacid (see WO 2006/136460 A2) were dissolved in 22 mL DMF. To thesolution were added 434 mg (3.36 mmol, 3.6 eq.)N,N-diisopropylethylamine and 1.28 g (3.36 mmol, 3.6 eq.) HATU. Theresulting reaction mixture was stirred for 2 hours at room temperature.After dropwise adding of a solution of 96.1 mg (0.93 mmol, 1eq.)N-(2-aminoethyl)ethane-1,2-diamine and of 434 mg (3.36 mmol, 3.6eq.) N,N-diisopropylethylamine in 9 mL DMF, the resulting reactionmixture was heated under stirring for 3 hours at 70° C. After coolingand diluting with dichloromethane, the solution was washed with aqueoussodium hydroxide (0.1 M), aqueous citric acid (1%), and water and wasdried over sodium sulfate. The crude product was isolated by evaporationunder reduced pressure and was purified by silica gel chromatographyyielding 451 mg (0.45 mmol, 48%) of the title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ=1.37 (s, 54H), 2.36-2.49 (m, 3H), 2.81-3.30(m, 17H), 3.36-3.70 (m, 3H), 6.16-6.92 (m, 6H), 7.77-8.35 (m, 2H) ppm.

UPLC (ACN-HCOOH): Rt.=1.49 min.

MS (ES⁺): m/z=1004.6 (M+H)⁺.

Example 2b3-Amino-N,N-bis(2-{[3-amino-2-(aminomethyl)propanoyl]amino}ethyl)-2-(aminomethyl)-propanamidehexahydrochloride

581 mg (0.58 mmol) Tert-butyl(12-{2-[(3-[(tert-butoxycarbonyl)amino]-2-{[(tert-butoxy-carbonyl)amino]methyl}propanoyl)amino]ethyl}-7,14-bis{[(tert-butoxycarbonyl)amino]methyl}-2,2-dimethyl-4,8,13-trioxo-3-oxa-5,9,12-triazapentadecan-1511)carbamate(example 2a) were dissolved in 9.3 mL methanol and 2.9 mL aqueoushydrochloric acid (37%). The reaction mixture was heated under stirringfor 2 hours at 50° C. For isolation, the suspension was evaporated todryness yielding 376 mg (0.60 mmol, 103%) of the title compound.

¹H-NMR (400 MHz, D₂O): δ=3.13-3.27 (m, 2H), 3.28-3.85 (m, 21H) ppm.

UPLC (ACN-HCOOH): Rt.=0.19 min.

MS (ES⁺): m/z=404.3 (M+H)⁺, free base.

Example 2 Hexagadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,10,15,19,22-hexaoxo-23-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,16-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-amino}methyl)-11-(2-{[3-{[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl]propanoyl}amino)acetyl]amino}-2-({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)propanoyl]-amino}ethyl)-4,7,11,14,18,21-hexaazatetracosan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

150 mg (241 μmol, 1 eq.)3-Amino-N,N-bis(2-{[3-amino-2-(aminomethyl)propanoyl]amino}-ethyl)-2-(aminomethyl)propanamidehexahydrochloride (example 2b) were dissolved in 60 mL DMSO. Afteradding of 467 mg (3.62 mmol, 15 eq.) N,N-diisopropylethylamine and 3.80g (5.06 mmol, 21 eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001/051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 15-20 mL. The concentrate waspoured under stirring in 400 mL ethyl acetate, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 677 mg (166 μmol, 69%) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.44 min.

MS (ES⁺): m/z (z=3)=1357.4 (M+3H)³⁺, m/z (z=4)=1018.8 (M+4H)⁴⁺], m/z(z=5)=815.7 (M+5H)⁵⁺.

Example 3 Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,12,15-tetraoxo-16-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

225 mg (1.65 mmol, 1 eq.) 2,2-Bis(aminomethyl)propane-1,3-diamine (seeW. Hayes et al., Tetrahedron 59 (2003), 7983-7996) were dissolved in 240mL DMSO. After addition of 1.71 g (13.2 mmol, 8 eq.)N,N-diisopropylethylamine and 14.9 g (19.85 mmol, 12 eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001/051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 40-50 mL. The concentrate waspoured under stirring in 600 mL ethyl acetate, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 3.42 g (80%, 1.33 mmol) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.42 min.

MS (ES⁺): m/z (z=2)=1290.4 (M+H)²⁺, m/z (z=3)=860.7 (M+H)³⁺.

Example 3 comprises a mixture of stereoisomers, which exhibit thefollowing absolute configurations: all-R, all-S, RRRS, SSSR, RRSS.

Example 3-1 Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)-acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclo-dodecan-1-yl}acetate

Example 3-1a Tert-butyl{10,10-bis[({[(tert-butoxycarbonyl)amino]acetyl}amino)methyl]-2,2-dimethyl-4,7,13-trioxo-3-oxa-5,8,12-triazatetradecan-14-yl}carbamate

A mixture of 2,2-bis(aminomethyl)propane-1,3-diamine tetrahydrochloride(851 mg, 3.06 mmol, 1 eq.; see W. Hayes et al., Tetrahedron 59 (2003),7983-7996) in dichloromethane (50 mL) was treated withN,N-diisopropylethylamine (6.00 eq., 3.20 mL, 18.4 mmol) and2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)glycinate (CAS No.[3392-07-2]; 6.00 eq., 5.00 g, 18.4 mmol) and stirred at roomtemperature for 2.5 days. The reaction mixture was diluted with water,the formed precipitate filtered off and washed with water anddichloromethane. The precipitated material was subjected to silica gelchromatography (dichloromethane/methanol) to give the title compound(800 mg, 34%).

¹H-NMR (400 MHz, DMSO-d₆): δ=1.36 (s, br, 36H), 2.74-2.76 (m, 8H),3.48-3.50 (m, 8H), 6.96 (s, br, 0.4H*), 7.40-7.42 (m, 3.6H*), 7.91-8.00(m, 4H) ppm.

LC-MS (ES⁺): m/z=761.4 (M+H)⁺; Rt.=1.16 min.

Example 3-1b2-Amino-N-(3-[(aminoacetyl)amino]-2,2-bis({[(aminoacetyl)amino]methyl}propyl)acetamidetetrahydrochloride

A suspension of tert-butyl{10,10-bis[({[(tert-butoxycarbonyl)amino]acetyl}amino)methyl]-2,2-dimethyl-4,7,13-trioxo-3-oxa-5,8,12-triazatetradecan-14-yl}carbamate(1.00 eq., 800 mg, 1.05 mmol) from example 11a in CPME (10 mL) wascooled to 0° C. and treated dropwise with HCl in CPME (10 eq., 3.5 mL ofa 3 M solution, 10.5 mmol). The reaction mixture was stirred at 0° C.for 1 h and at rt overnight upon which dioxane (4 mL) and another amountof HCl in CPME (30 eq., 11 mL of a 3 M solution, 32 mmol) were added andstirring at rt continued for 2 days. The resulting suspension wasconcentrated in vacuo to give the title compound (575 mg, quant.) whichwas not further purified.

¹H-NMR (400 MHz, DMSO-d₆): δ=3.17-3.18 (m, 8H), 3.59-3.61 (m, 8H), 8.21(s, br, 12H), 8.55 (t, 4H) ppm.

LC-MS (ES⁺): m/z=361.2 (M−3HCl−Cl⁻)⁺; Rt.=0.10 min.

Example 3-1c Benzyl (2S)-2-{[(trifluoromethyl)sulfonyl]oxy}propanoate

Prepared according to H. C. J. Ottenheim et al., Tetrahedron 44 (1988),5583-5595: A solution of (S)-(−)-lactic acid benzyl ester (CAS No.[56777-24-3]; 1.00 eq., 5.00 g, 27.7 mmol) in dry dichloromethane (95mL) was cooled to 0° C. and treated with trifluoromethanesulfonicanhydride (CAS No. [358-23-6]; 1.1 eq., 5.2 mL, 8.6 g, 31 mmol). Afterstirring for 5 min, 2,6-dimethylpyridine (1.15 eq., 3.72 mL, 3.42 g) wasadded and stirring continued for another 5 min. The obtained reactionmixture was directly used in the next step.

Example 3-1d Benzyl(2R)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoate

A solution of tri-tert-butyl2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (CAS No.[122555-91-3]; 1.00 eq., 9.52 g, 18.5 mmol) in dry dichloromethane (75mL) was cooled to 0° C. and treated with the reaction mixture of benzyl(2S)-2-{[(trifluoromethyl)sulfonyl]oxy}propanoate in dichloromethaneprepared in example 3-1c; and N,N-diisopropylethylamine (3.0 eq, 9.7 mL,55 mmol). The resulting solution was stirred at rt for 6 days upon whichit was diluted with ethyl acetate and washed with saturated aqueoussodium bicarbonate. The organic layer was dried over sodium sulfate andconcentrated under reduced pressure. The obtained material was purifiedby amino phase silica gel chromatography (KP-NH®, hexane/ethyl acetateto dichloromethane/methanol) to give the title compound (1.92 g, 14%).

¹H-NMR (400 MHz, DMSO-d₆): δ=1.20 (d, 3H), 1.37-1.45 (m, 27H), 1.98-2.01(m, 3H), 2.08-2.24 (m, 5H), 2.57-2.84 (m, 7H), 2.94-3.11 (m, 4H),3.38-3.48 (m, 3H), 3.75 (q, 1H), 5.07-5.17 (m, 2H), 7.32-7.40 (m, 5H)ppm.

LC-MS (ES⁺): m/z=677.5 (M+H)⁺, m/z (z=2)=339.2 (M+H)²⁺; Rt.=1.06 min.

Example 3-1e(2R)-2-[4,7,10-Tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-propanoicacid

A solution of benzyl(2R)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoate(example 3-1d; 1.92 g, 2.84 mmol) in methanol (17.5 mL) was treated withPd/C (10 wt %; 0.050 eq., 151 mg, 0.14 mmol) and stirred under ahydrogen atmosphere at room temperature for 20 hours. The reactionmixture was filtrated over Celite®, washed with methanol, and thefiltrate concentrated in vacuo to give the title compound (1.51 g, 88%)which was not further purified.

¹H-NMR (400 MHz, DMSO-d₆): δ=1.11 (s, br, 3H), 1.42-1.43 (m, 27H),1.97-2.13 (m, 5H), 2.56-2.82 (m, 7H), 2.97-3.07 (m, 4H), 3.34-3.53 (m,7H), 12.8 (s, br, 1H) ppm.

UPLC (ACN-NH₃): Rt.=1.31 min.

MS (ES⁺): m/z=587 (M+H)⁺.

LC-MS (ES⁺): m/z=587 (M+H)⁺, m/z (z=2)=294.2 (M+H)²⁺; Rt.=0.79 min.

Example 3-1f Tert-butyl{4,10-bis(2-tert-butoxy-2-oxoethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}-amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclo-dodecan-1-yl}acetate

A mixture of(2R)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoic acid (example 3-1e; 12.0 eq., 1.50 g, 2.56 mmol)in N,N-dimethylacetamide (15 mL) was treated with HATU (14.4 eq., 1.17g, 3.07 mmol) and N,N-diisopropylethylamine (14.4 eq., 534 μL, 3.07mmol) and stirred at rt for 20 minutes. A suspension of2-amino-N-(3-[(aminoacetyl) amino]-2,2-bis{[(aminoacetyl)amino]methyl}propyl)acetamide tetrahydrochloride (example 3-1b; 1.00 eq., 108 mg, 213μmol) in N,N-dimethylacetamide (6 mL) was added and the resultingmixture stirred at 50° C. overnight. The reaction mixture wasconcentrated under reduced pressure and the obtained residue subjectedto amino phase silica gel chromatography (KP-NH®, ethyl acetate to ethylacetate/methanol) to give the title compound (260 mg, 42%).

¹H-NMR (400 MHz, DMSO-d₆): δ=1.03 (s, br, 5H), 1.28 (s, br, 7H),1.36-1.43 (m, 108H), 1.87-2.24 (m, 23H), 2.42 (s, br, 4H), 2.53-2.84 (m,41H), 2.97-3.18 (m, 17H), 3.28 (s, br, 5H), 3.39-3.46 (m, 6H), 3.58 (s,br, 7H), 3.76 (s, br, 2H), 4.01 (s, br, 3H), 7.81 (s, br, 5H), 8.33 (s,br, 2H), 9.27 (s, br, 1H) ppm.

UPLC (ACN-NH₃): Rt.=1.23 min.

MS (ES⁺): m/z (z=4)=660 (M+H)⁴⁺.

LC-MS (ES⁺): m/z (z=2)=1318 (M+H)²⁺, m/z (z=3)=879 (M+H)³⁺, m/z(z=4)=660 (M+H)⁴⁺; Rt.=0.94 min.

Example 3-1g{4,10-Bis(carboxymethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}aceticacid

Tert-butyl{4,10-bis(2-tert-butoxy-2-oxoethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate(example 3-1f; 260 mg, 0.099 mmol) was treated with TFA (25 mL) understirring at room temperature overnight. The reaction mixture wasconcentrated under reduced pressure, the obtained residue taken up withwater (20 mL) and lyophilized. The crude product was used withoutfurther characterization in the next chemical step.

Example 3-1 Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)-acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclo-dodecan-1-yl}acetate

The crude material{4,10-bis(carboxymethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetic acid from example 3-1g was dissolved in water (20 mL).Tris(acetato-kappaO)gadolinium tetrahydrate (298 mg, 0.734 mmol) wasadded and the reaction mixture stirred at 70° C. for 2 h. The pH valueof the resulting solution was adjusted to 4.5 by addition of aqueoussodium hydroxide solution (2 N) and stirring at 70° C. continued for 2days. The resulting solution was ultrafiltered with water (7×100 mL)using a 1 kDa membrane and the final retentate was lyophilized yieldingthe title compound (70 mg, 27% over two steps).

UPLC (ACN-HCOOH): Rt.=0.39 min.

MS (ES⁺): m/z (z=2)=1290.1 (M+H)²⁺, m/z (z=3)=860.3 (M+H)³⁺.

LC-MS (ES⁺): m/z (z=2)=1290.3 (M+H)²⁺, m/z (z=3)=860.9 (M+H)³⁺, m/z(z=4)=645.6 (M+H)⁴⁺; Rt.=0.25 min.

Example 3-2 Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(25,165)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}-acetate

Example 3-2a Benzyl (2R)-2-{[(trifluoromethyl)sulfonyl]oxy}propanoate

Prepared in analogy to the corresponding S-isomer (example 3-1c) from(R)-(+)-lactic acid benzyl ester (CAS No. [74094-05-6]; 8.00 g, 44.4mmol) in dichloromethane. The obtained reaction mixture was directlyused in the next step.

Example 3-2b Benzyl(2S)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoate

Prepared in analogy to the corresponding R-isomer (example 3-1d) fromtri-tert-butyl2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (CAS No.[122555-91-3]; 1.00 eq., 15.2 g, 29.6 mmol) and the reaction mixture ofbenzyl (2R)-2-{[(trifluoromethyl) sulfonyl]oxy}propanoate indichloromethane prepared in example 3-2a.

LC-MS (ES⁺): m/z=677.4 (M+H)⁺, m/z (z=2)=339.2 (M+H)²⁺; Rt.=0.94 min.

Example 3-2c(2S)-2-[4,7,10-Tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-propanoicacid

Prepared in analogy to the corresponding R-isomer (example 3-1e) frombenzyl(2S)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoate(example 3-2b).

UPLC (ACN-NH₃): Rt.=1.31 min.

MS (ES⁺): m/z=587 (M+H)⁺.

LC-MS (ES⁺): m/z=587.4 (M+H)⁺, m/z (z=2)=294.2 (M+H)²⁺; Rt.=0.82 min.

Example 3-2d Tert-butyl{4,10-bis(2-tert-butoxy-2-oxoethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}-amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraaza-cyclododecan-1-yl}acetate

Prepared in analogy to the corresponding R-isomer (example 3-1f) from(2S)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoicacid (example 3-2c) and 2-amino-N-(3-[(aminoacetyl)amino]-2,2-bis{[(aminoacetylamino]methyl} propyl)acetamidetetrahydrochloride (example 3-1b).

LC-MS (ES⁺): m/z (z=2)=1318 (M+H)²⁺, m/z (z=3)=879 (M+H)³⁺, m/z(z=4)=660 (M+H)⁴⁺; Rt.=0.95 min.

Example 3-2e{4,10-Bis(carboxymethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}aceticacid

Prepared in analogy to the corresponding R-isomer (example 3-1g) fromtert-butyl{4,10-bis(2-tert-butoxy-2-oxoethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate(example 3-2d). The crude product was used without furthercharacterization in the next chemical step.

Example 3-2 Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}-acetate

Prepared in analogy to the corresponding R-isomer (example 3-1) from{4,10-bis(carboxymethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-cyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}aceticacid (example 3-2e) and tris(acetato-kappaO)gadolinium tetrahydrate atpH 4.5. The resulting reaction solution was ultrafiltered with water(8×100 mL) using a 1 kDa membrane and the final retentate lyophilizedand purified by preparative HPLC.

UPLC (ACN-HCOOH): Rt.=0.41 min.

MS (ES⁺): m/z (z=2)=1290 (M+H)²⁺, m/z (z=3)=861 (M+H)³⁺.

LC-MS (ES⁺): m/z (z=2)=1290 (M+H)²⁺, m/z (z=3)=860 (M+H)³⁺, m/z(z=4)=645.6 (M+H)⁴⁺; Rt.=0.23 min.

Example 4 Pentagadolinium[4-(1-{[2-(bis{2-[({1,4-bis[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-1,4-diazepan-6-yl}carbonyl)-amino]ethyl}amino)-2-oxoethyl]amino}-1-oxopropan-2-yl)-7,10-bis(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetate

Example 4a 6-(Methoxycarbonyl)-1,4-diazepanediium dichloride

6.00 g (17.7 mmol) Methyl 1,4-dibenzyl-1,4-diazepane-6-carboxylate [seeU.S. Pat. No. 5,866,562] were dissolved in 30 mL methanol. After addingof 6 mL aqueous hydrochloric acid (37%), 6 mL water and 600 mg palladiumon charcoal (10%), the reaction mixture was hydrogenated (1 atm) for 17hours at 40° C. The catalyst was filtered off and the solution wasevaporated under reduced pressure yielding 4.1 g (17.7 mmol, 100%) ofthe title compound.

¹H-NMR (400 MHz, D₂O): δ=3.62-3.84 (m, 9H), 3.87 (s, 3H) ppm.

UPLC (ACN-HCOOH): Rt.=0.20 min.

MS (ES⁺): m/z=159.1 (M+H)⁺, free base.

Example 4b 1,4-Di-tert-butyl 6-methyl 1,4-diazepane-1,4,6-tricarboxylate

4.00 g (17.3 mmol, 1 eq.) 6-(Methoxycarbonyl)-1,4-diazepanediiumdichloride (example 4a) were dissolved in 80 mL DMF. After addition of7.71 g (76.2 mmol, 4.4 eq.) trimethyl amine and 8.31 g (38.1 mmol, 2.2eq.) di-tert-butyl dicarbonate, the resulting reaction mixture wasstirred overnight at room temperature. The suspension was filtered, thefiltrate evaporated under reduced pressure and diluted with ethylacetate. The resulting solution was washed with aqueous citric acid(pH=3-4), half saturated aqueous sodium bicarbonate, was dried oversodium sulfate, and evaporated under reduced pressure yielding 4.92 g(13.7 mmol, 79%) of the title compound.

¹H-NMR (300 MHz, DMSO-d₆): δ=1.36 (s, 18H), 2.69-3.27 (m, 4H), 3.35-4.00(m, 5H), 3.62 (s, 3H) ppm.

UPLC (ACN-HCOOH): Rt.=1.32 min.

MS (ES⁺): m/z=359.2 (M+H)⁺.

Example 4c 1,4-Bis(tert-butoxycarbonyl)-1,4-diazepane-6-carboxylic acid

4.86 g (13.66 mmol) 1,4-Di-tert-butyl 6-methyl1,4-diazepane-1,4,6-tricarboxylate (example 4b) were dissolved in 82 mLTHF. After adding of 27 mL aqueous sodium hydroxide (2 M), the resultingreaction mixture was stirred for 20 hours at room temperature, wasdiluted with water, and was acidified (pH=3-4) by addition of citricacid. The crude product was extracted with dichloromethane, the organiclayer was washed with brine, dried over sodium sulfate, and wasevaporated to dryness yielding 4.67 g (12.4 mmol, 91%) of the titlecompound.

¹H-NMR (400 MHz, DMSO-d₆): δ=1.38 (s, 18H), 2.58-2.86 (m, 1H), 2.94-4.00(m, 8H), 12.50 (s, br, 1H) ppm.

UPLC (ACN-HCOOH): Rt.=1.12 min.

MS (ES⁺): m/z=345.2 (M+H)⁺.

Example 4d Di-tert-butyl6-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}-1,4-diazepane-1,4-dicarboxylate

1.76 g (5.11 mmol, 1 eq.)1,4-Bis(tert-butoxycarbonyl)-1,4-diazepane-6-carboxylic acid (example4c) and 0.65 g (5.62 mmol, 1.1 eq.) 1-hydroxypyrrolidine-2,5-dione weredissolved in 50 mL THF. A solution of 1.16 g (5.62 mmol, 1.1 eq.)N,N′-dicyclohexylcarbodiimide in 30 mL THF was added and the resultingreaction mixture was refluxed for 5 hours. The suspension was cooled to0° C. and the precipitated u rea was filtered off. The final solution ofthe activated ester was directly used for the next chemical step.

UPLC (ACN-HCOOH): Rt.=1.24 min.

MS (ES⁺): m/z=442.3 (M+H)⁺.

Example 4e Tetra-tert-butyl6,6′-[iminobis(ethane-2,1-diylcarbamoyl)]bis(1,4-diazepane-1,4-dicarboxylate)

To the solution of the activated ester (5.11 mmol, 2.2 eq.)di-tert-butyl6-{[(2,5-dioxo-pyrrolidin-1-yl)oxy]carbonyl}-1,4-diazepane-1,4-dicarboxylatefrom example 4d were added 517 mg (5.11 mmol, 2.2 eq.) triethylamine and240 mg (2.32 mmol, 1 eq.)N-(2-aminoethyl)ethane-1,2-diamine. Theresulting reaction mixture was stirred for 20 hours at room temperatureand was diluted with dichloromethane. The solution was washed withaqueous sodium hydroxide (0.1 M), then with water, and was dried oversodium sulfate. The crude product was isolated by evaporation and waspurified by silica gel chromatography yielding 1.20 g (1.59 mmol, 68%)of the title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ=1.37 (s, 36H), 2.51-2.70 (m, 7H), 2.85-3.28(m, 12H), 3.45-4.10 (m, 8H), 7.69-8.27 (m, 2H) ppm.

UPLC (ACN-HCOOH): Rt.=1.20 min.

MS (ES⁺): m/z=756.7 (M+H)⁺.

Example 4f N,N′-(Iminodiethane-2,1-diyl)bis(1,4-diazepane-6-carboxamide)pentahydrochloride

385 mg (0.51 mmol) Tetra-tert-butyl6,6′-[iminobis(ethane-2,1-diylcarbamoyl)]bis(1,4-diazepane-1,4-dicarboxylate)(example 4e) were dissolved in 5.7 mL methanol and 1.7 mL aqueoushydrochloric acid (37%). The reaction mixture was heated under stirringfor 2 hours at 50° C. For isolation the suspension was evaporate d todryness yielding 277 mg (0.51 mmol, 100%) of the title compound.

¹H-NMR (400 MHz, D₂O): δ=3.18 (t, 4H), 3.32-3.40 (m, 2H), 3.51 (t, 4H),3.57-3.69 (m, 16H) ppm.

UPLC (ACN-HCOOH): Rt.=0.24 min.

MS (ES⁺): m/z=356.3 (M+H)⁺, free base.

Example 4 Pentagadolinium[4-(1-{[2-(bis{2-[({1,4-bis[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]-1,4-diazepan-6-yl}carbonyl)-amino]ethyl}amino)-2-oxoethyl]amino}-1-oxopropan-2-yl)-7,10-bis(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetate

150 mg (279 μmol, 1 eq.)N,N′-(Iminodiethane-2,1-diyl)bis(1,4-diazepane-6-carboxamide)pentahydrochloride (example 4f) were dissolved in 60 mL DMSO. Afteraddition of 451 mg (3.49 mmol, 12.5 eq.), N,N-diisopropylethylamine and3.67 g (4.88 mmol, 17.5 eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 15-20 mL. The concentrate waspoured under stirring in 400 mL ethyl acetate, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 672 mg (197 μmol, 70%) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.43 min.

MS (ES⁻): m/z (z=2)=1706.3 (M−2H)²⁻ m; (ES⁺): m/z (z=4)=854.5 (M+4H)⁴⁺.

Example 5 Hexagadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-{ethane-1,2-diylcarbamoyl-1,4-diazepane-6,1,4-triyltris[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}octadecaacetate

Example 5a Hexa-tert-butyl6,6′,6″-(ethane-1,2-diylcarbamoyl)tris(1,4-diazepane-1,4-dicarboxylate)

1.20 g (3.48 mmol, 3 eq.)1,4-Bis(tert-butoxycarbonyl)-1,4-diazepane-6-carboxylic acid (example4c), 540 mg (4.18 mmol, 3.6 eq.) diisopropylethylamine and 1.59 g (4.18mmol, 3.6 eq.) HATU were dissolved in 30 mL DMF and stirred for 2 hoursat room temperature. After drop wise addition of a solution of 120 mg(1.16 mmol, 1 eq.), N-(2-aminoethyl)ethane-1,2-diamine and of 540 mg(4.18 mmol, 3.6 eq.) N,N-diisopropylethylamine in 8 mL DMF, theresulting reaction mixture was heated under stirring for 3 hours at 70°C. After cooling and diluting with dichloromethane, the solution waswashed with aqueous sodium hydroxide (0.1 M), with aqueous citric acid(1%), with water and was dried over sodium sulfate. The crude productwas isolated by evaporation under reduced pressure and was purified bysilica gel chromatography yielding 660 mg (0.61 mmol, 52%) of the titlecompound.

¹H-NMR (400 MHz, DMSO-d₆): δ=1.38 (s, 54H), 2.55-4.06 (m, 35H),7.90-8.52 (m, 2H) ppm.

UPLC (ACN-HCOOH): Rt.=1.64 min.

MS (ES⁺): m/z=1082.7 (M+H)⁺.

Example 5bN,N-Bis{2-[(1,4-diazepan-6-ylcarbonyl)amino]ethyl}-1,4-diazepane-6-carboxamidehexahydrochloride

654 mg (0.60 mmol) Hexa-tert-butyl6,6′,6″-(ethane-1,2-diylcarbamoyl)tris(1,4-diazepane-1,4-dicarboxylate)(example 5a) were dissolved in 6.8 mL methanol and 3 mL aqueoushydrochloric acid (37%). The reaction mixture was heated under stirringfor 2.5 hours at 50° C. For isolation, the suspension was evaporated todryness yielding 441 mg (0.63 mmol, 105%) of the title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ=3.20-3.71 (m, 35H), 8.50-8.80 ppm (m, 2H),9.76 (s, br, 12H).

UPLC (ACN-HCOOH): Rt.=0.19 min.

MS (ES⁺): m/z=482.3 (M+H)⁺, free base.

Example 5 Hexagadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-{ethane-1,2-diylcarbamoyl-1,4-diazepane-6,1,4-triyltris[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}octadecaacetate

150 mg (214 μmol, 1 eq.)N,N-Bis{2-[(1,4-diazepan-6-ylcarbonyl)amino]ethyl}-1,4-diazepane-6-carboxamidehexahydrochloride (example 5b) were dissolved in 60 mL DMSO.

After adding of 0.42 g (3.21 mmol, 15 eq.), N,N-diisopropylethylamineand 3.38 g (4.50 mmol, 21 eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001/051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 15-20 mL. The concentrate waspoured under stirring in 400 mL ethyl acetate, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 595 mg (143 μmol, 67%) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.41 min.

MS (ES⁺): m/z (z=3)=1384.6 (M+H)³⁺, m/z (z=4)=1039.5 (M+H)⁴⁺, m/z(z=5)=831.6 (M+H)⁵⁺.

Example 6 Hexagadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-(1,4,7-triazonane-1,4,7-triyltris{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})octadecaacetate

Example 6a Hexa-tert-butyl6,6′,6″-(1,4,7-triazonane-1,4,7-triyltricarbonyl)tris(1,4-diazepane-1,4-dicarboxylate)

800 mg (2.32 mmol, 3 eq.)1,4-Bis(tert-butoxycarbonyl)-1,4-diazepane-6-carboxylic acid (example4c), 360 mg (2.79 mmol, 3.6 eq.) diisopropylethylamine and 1.06 g (2.79mmol, 3.6 eq.) HATU were dissolved in 20 mL DMF and stirred for 2 hoursat room temperature. After dropwise adding of a solution of 100 mg (774μmol, 1 eq.) 1,4,7-triazonane trihydrochloride and of 360 mg (2.79 mmol,3.6 eq.) N,N-diisopropylethylamine in 5 mL DMF, the resulting reactionmixture was heated under stirring for 3 hours at 70° C. After coolingand diluting with dichloromethane, the solution was washed with aqueoussodium hydroxide (0.1 M), with aqueous citric acid (1%), with water andwas dried over sodium sulfate. The crude product was isolated byevaporation under reduced pressure and was purified by silica gelchromatography yielding 545 mg (492 μmol, 63%) of the title compound.

¹H-NMR (400 MHz, CDCl₃): δ=1.47 (s, 54H), 2.85-4.45 (m, 39H) ppm.

UPLC (ACN-HCOOH): Rt.=1.73 min.

MS (ES⁺): m/z=1108.8 (M+H)⁺.

Example 6b 1,4,7-Triazonane-1,4,7-triyltris(1,4-diazepan-6-ylmethanone)hexahydrochloride

380 mg (343 μmop Hexa-tert-butyl6,6′,6″-(1,4,7-triazonane-1,4,7-triyltricarbonyl)tris(1,4-diazepane-1,4-dicarboxylate)(example 6a) were dissolved in 3.90 mL methanol and 1.72 mL aqueoushydrochloric acid (37%). The reaction mixture was heated under stirringfor 2.5 hours at 50° C. For isolation the suspension was evaporated todryness yielding 257 mg (354 μmol, 103%) of the title compound.

UPLC (ACN-HCOOH): Rt.=0.19 min.

MS (ES⁺): m/z=508.4 (M+H)⁺, free base.

Example 6 Hexagadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-(1,4,7-triazonane-1,4,7-triyltris{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})octadecaacetate

175 mg (241 μmol, 1 eq.)1,4,7-Triazonane-1,4,7-triyltris(1,4-diazepan-6-ylmethanone)hexahydrochloride (example 6b) were dissolved in 60 mL DMSO. Afteradding of 467 mg (3.61 mmol, 15 eq.) N,N-diisopropylethylamine and 3.80g (5.06 mmol, 21 eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 15-20 mL. The concentrate waspoured under stirring in 400 mL ethyl acetate, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 590 mg (141 μmol, 58%) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.43 min.

MS (ES⁺): m/z (z=3)=1393.1 (M+3H)³⁺, m/z (z=4)=1045.5 (M+4H)⁴⁺, m/z(z=5)=837.0 [(M+5H)⁵⁺.

Example 7 Tetragadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-{1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetrayltetrakis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}dodecaacetate

35 mg (203 μmol, 1 eq.) 1,4,7,10-Tetraazacyclododecane were dissolved in60 mL DMSO. After adding of 2.14 g (2.84 mmol, 14 eq.) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate(see WO 2001051095 A2), the resulting reaction mixture was stirred andheated for 8 hours at 50° C. The cooled solution was concentrated underreduced pressure to a final volume of 15-20 mL. The concentrate waspoured under stirring in 400 mL ethyl acetate, the formed precipitatewas filtered off and was dried in vacuo. The solid was dissolved inwater, the resulting solution was ultrafiltered with water using a 1 kDamembrane and the final retentate was lyophilized. The crude product waspurified by RP-chromatography yielding 28 mg (10.6 μmol, 5%) of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.41 min.

MS (ES⁺): m/z (z=2)=1311.7 (M+2H)²⁺, m/z (z=3)=873.1 (M+3H)³⁺.

Example 8 Hexagadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-{3,7,10-triazatricyclo[3.3.3.0^(1,5)]undecane-3,7,10-triyltris[carbonyl(3,6,11,14-tetraoxo-4,7,10,13-tetraazahexadecane-8,2,15-triyl)di-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}octadecaacetate

Example 8aTetrahydro-1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrole

4.0 g (6.5 mmol)2,5,8-Tris((4-methylphenyl)sulfonyl)tetrahydro-1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrole(prepared via the procedures outlined in J. Org. Chem. 1996, 61,8897-8903) was refluxed in 44 mL aqueous hydrobromic acid (47%) and 24mL acetic acid for 18 hours. The solvent was removed in vacuo, theresidue dissolved in water, and the aqueous phase was washed two timeswith dichloromethane. The aqueous phase was lyophilized and taken up ina small amount of water and passed through an anionic exchange column(DOWEX 1×8) by elution with water. The basic fraction was collected andconcentrated to yield 0.89 g oftetrahydro-1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrole asfree base.

¹H-NMR (400 MHz, D₂O): δ=2.74 (s, 12H) ppm.

Example 8bTert-butyl-{1-[5,8-bis{2,3-bis[(tert-butoxycarbonyl)amino]propanoyl}dihydro-1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrol-2(3H)-yl]-3-[(tert-butoxycarbonyl)amino]-1-oxopropan-2-yl}carbamate

A solution prepared from 431.5 mg (0.89 mmol, CAS[201472-68-6])N-(tert-butoxycarbonyl)-3-[(tert-butoxycarbonyl)amino]alanineN,N-dicyclohexylammonium salt, 0.44 mL (2.54 mmol)N,N-diisopropylethylamine and 386 mg (1.0 mmol) HATU in 4.3 mL DMF wasadded to 38.9 mg (254 μmol) oftetrahydro-1H,4H-3a,6a-(methanoimino-methano)pyrrolo[3,4-c]pyrrole in 2mL DMF. After stirring the combined mixture for 20 min at roomtemperature, the solvent was removed in vacuo and the residue purifiedby chromatography on amino phase silica gel (ethyl acetate in hexane, 0to 100%) followed by preparative HPLC (C18-Chromatorex 10 μm,acetonitrile in water+0.1% formic acid, 65% to 100%) to yield 68.6 mg oftert-butyl-{1-[5,8-bis{2,3-bis[(tert-butoxycarbonyl)amino]propanoyl}dihydro-1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrol-2(3H)-yl]-3-[(tert-butoxycarbonyl)amino]-1-oxopropan-2-yl}carbamate.

¹H-NMR (300 MHz, CDCl₃): δ=1.43 s, br, 54H), 3.34-3.97 (m, 18H), 4.48(s, br, 3H), 5.01-5.67 (m, 6H) ppm.

UPLC (ACN-HCOOH): Rt.=1.48 min.

MS (ES⁺): m/z=1012.6 (M+H)⁺.

Example 8c3,3′,3″-[1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrole-2,5,8(3H,6H)-triyl]-tris(3-oxopropane-1,2-diaminium)hexachloride

65 mg (60 μmopTert-butyl-{1-[5,8-bis{2,3-bis[(tert-butoxycarbonyl)amino]propanoyl}dihydro-1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrol-2(3H)-yl]-3-[(tert-butoxy-carbonyl)amino]-1-oxopropan-2-yl}carbamate(example 8b) were dissolved in 2.0 mL DMF and 0.48 mL hydrochloric acidin dioxane (4 M, 0.19 mmol) were added. The reaction mixture was heatedunder microwave radiation for 10 min at 80° C. while stirring. Thesolvent was removed in vacuo, the residue taken up in a small amount ofwater and lyophilized to yield 38.9 mg of3,3′,3″-[1H,4H-3a,6a-(methanoiminomethano)pyrrolo[3,4-c]pyrrole-2,5,8(3H,6H)-triyl]tris(3-oxopropane-1,2-diaminium)hexachloride.

¹H-NMR (600 MHz, D₂O): δ=3.40-3.50 (m, 3H), 3.52-3.56 (m, 3H), 3.79-4.19(m, 12H), 4.51-4.54 (m, 3H) ppm.

UPLC (ACN-HCOOH): Rt.=0.20 min.

MS (ES⁺): m/z=412.3([M+H)⁺, free base.

Example 8 Hexagadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″,2″″″″″″,2′″″″″″″,2″″″″″″″,2′″″″″″″″,2″″″″″″″″,2′″″″″″″″″-{3,7,10-triazatricyclo[3.3.3.0^(1,5)]undecane-3,7,10-triyltris[carbonyl(3,6,11,14-tetraoxo-4,7,10,13-tetraazahexadecane-8,2,15-triyl)di-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}octadecaacetate

30 mg (48 μmop3,3′,3″-1H,4H-3a,6a-(Methanoiminomethano)pyrrolo[3,4-c]pyrrole-2,5,8(3H,6H)-triyl]tris(3-oxopropane-1,2-diaminium) hexachloride (example 8c)were dissolved in a mixture of 1.8 mL DMSO, 1.8 mL DMF, and 116 μLpyridine. At 60° C. 281 mg (0.38 mmol, WO 2001051095 A2) of gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetatewere added followed by 44 μL trimethylamine and the resulting reactionmixture was stirred for 15 hours at 60° C. and at room temperature fortwo days. Another amount of gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetate (56 mg, 75 μmop and trimethylamine (5.4μL) was added at 60° C. and stirring at 60° C. was continued for 15 hours. The solvent was removed in vacuo, the residue taken up in 200 mL ofwater, and the resulting solution was ultrafiltered using a 1 kDamembrane. After diluting the retentate two times with additional 200 mLof deionized water and continuing the ultrafiltration, the finalretentate was lyophilized. The residue was dissolved in a mixture of 1.6mL DMSO, 1.6 mL DMF, and 105 μL pyridine and addition of 261 mg (0.35mmol) gadolinium2,2′,2″-[10-(1-{[2-(4-nitrophenoxy)-2-oxoethyl]amino}-1-oxopropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl]triacetateand 48 μL triethylamine at 60° C. was repeated a third time. Afterstirring for 18 hours at 60° C. the ultrafiltration procedure using a 1kDa membrane was repeated and the retentate after three 200 mLfiltrations was lyophilized. The crude product was purified bypreparative HPLC (XBrigde C18, 5 μm, acetonitrile in water +0.1% formicacid, 0% to 7%) to yield 51 mg of the title compound.

UPLC (ACN-HCOOH long run): Rt.=2.95 min.

MS (ES+): m/z (z=3)=1360.4 (M+3H)³⁺, m/z (z=4)=1021.3 (M+4H)⁴⁺, m/z(z=5)=817.5 (M+5H)⁵⁺.

Example 9

Tetragadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-(3,7,9-triazabicyclo[3.3.1]nonane-3,7-diylbis{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})dodecaacetate

Example 9a 3,7,9-Triazabicyclo[3.3.1]nonane

220 mg (0.49 mmol)3,9-Dibenzyl-7-(phenylsulfonyl)-3,7,9-triazabicyclo[3.3.1]nonane(prepared via the procedures outlined in Tetrahedron Lett., 2005, 46,5577-5580) was refluxed in 3.4 mL aqueous hydrobromic acid (47%) and 1.8mL acetic acid for 17 hours. The solvent was removed in vacuo, theresidue dissolved in water and the aqueous phase was washed two timeswith dichloromethane. The aqueous phase was lyophilized and taken up ina small amount of water and passed through an anionic exchange column(DOWEX 1×8) by elution with water. The basic fraction was collected andconcentrated to yield 29.6 mg of 3,7,9-triazabicyclo[3.3.1]nonane as thefree base.

¹H-NMR (400 MHz, D₂O): δ=2.88 (t, 2H), 3.15 (d, 8H) ppm.

Example 9b 6-(Methoxycarbonyl)-1,4-diazepinediium dichloride

To 8.3 g (24.5 mmol)methyl 1,4-dibenzyl-1,4-diazepane-6-carboxylate(prepared in analogy to U.S. Pat. No. 5,866,562, p. 9) in 42 mL methanolwere added 8.3 mL concentrated hydrochloric acid, 2 mL of water and 830mg palladium on charcoal (10%). The suspension was stirred under ahydrogen atmosphere for 5 hours at 40° C. and 17 hours at roomtemperature. The mixture was filtrated trough a path of Celite® and thefiltrate concentrated in vacuo upon which toluene was added two timesand removed in vacuo. The residue was dissolved in water and lyophilizedto yield 5.65 g of 6-(methoxycarbonyl)-1,4-diazepanediium dichloride.

¹H-NMR (400 MHz, D₂O): δ=3.49-3.68 (m, 9H), 3.70-3.73 (m, 4H), 3.75 (s,3H) ppm.

Example 9c Methyl1,4-bis{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,4-diazepane-6-carboxylate

To 200 mg (0.78 mmol) of 6-(methoxycarbonyl)-1,4-diazepanediiumdichloride in 10 mL dichloromethane were added 10 mL (6.2 mmol)N,N-diisopropylethylamine and the mixture stirred for 5 min at roomtemperature. 1.04 g (1.56 mmol) tri-tert-butyl2,2′,2″-(10-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (prepared in analogy to Cong Li et al., J. Am. Chem. Soc.2006, 128, p. 15072-15073; S3-5 and Galibert et al., Bioorg. Med. Chem.Letters 2010 (20), 5422-5425) was added and the mixture was stirred for18 hours at room temperature. The solvent was removed under reducedpressure and the residue was purified by chromatography on amino phasesilica gel (ethyl acetate in hexane, 20 to 100%, then ethanol in ethylacetate 0 to 100%) to yield 210 mg of the title compound.

UPLC (ACN-HCOOH): Rt.=0.94 min.

MS (ES⁺): m/z=1267.6 (M+1H)⁺

Example 9d Dodeca-tert-butyl2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-(3,7,9-triazabicyclo[3.3.1]nonane-3,7-diylbis{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})dodecaacetate

305 mg (0.24 mmol) Methyl1,4-bis{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,4-diazepane-6-carboxylate(example 9c) were dissolved in 3.9 mL THF and a solution of 6.6 mglithium hydroxide in 0.87 mL water was added. After stirring for 15 min,the solvent was removed under reduced pressure and the crude lithium1,4-bis{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,4-diazepane-6-carboxylate(300 mg) was dissolved in 2.0 mL dichloromethane. 120 μL (0.71 mmol)N,N-Diisopropylethylamine, 112 mg (0.30 mmol) HATU and 40 mg (0.30 mmol)3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol were added and after stirring for15 min a solution of 15 mg (0.12 mmol) of3,7,9-triazabicyclo[3.3.1]nonane in 1 mL dichloromethane was added andthe mixture was stirred for 3 days. To additional 170 mg of raw lithium1,4-bis{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,4-diazepane-6-carboxylatein 1 mL dichloromethane were added 67 mg (0.18 mmol) HATU, 24 mg (0.18mmol) 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol over 15 min and 50 μLN,N-diisopropylethylamine. After stirring for 15 minutes the freshlyprepared HATU solution was added to the reaction mixture. After one daythe solvent was removed under reduced pressure upon which toluene wasadded six times and removed in vacuo. The residue was purified bychromatography on amino phase silica gel (ethyl acetate in hexane, 0 to100%, then ethanol in ethyl acetate 0 to 40%) to yield 181 mg of thetitle compound.

UPLC (ACN-HCOOH): Rt.=0.78-0.84 min.

MS (ES⁻): m/z (z=2)=1298.7 (M−2H)²⁻

Example 9 Tetragadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-(3,7,9-triazabicyclo[3.3.1]nonane-3,7-diylbis{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxoethane-2,1-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})dodecaacetate

390 mg (mmol) Dodeca-tert-butyl2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-(3,7,9-triazabicyclo[3.3.1]nonane-3,7-diylbis{carbonyl-1,4-diazepane-6,1,4-triylbis[(2-oxo-ethane-2,1-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]})dodecaacetate(example 9d) were dissolved in 10.8 mL water and the solution wasadjusted to pH 2.5 by addition of aqueous hydrochloric acid (2M). 440 mg(1.25 mmol) Gadolinium(III)oxide were added and the mixture was stirredat 80° C. for 17 hours, while the pH of the suspension changed to pH 5.The mixture was diluted with water, sonicated and filtrated. Thefiltrate was ultrafiltered using a 1 kDa membrane. After diluting theretentate two times with additional 100 mL of deionized water andcontinuing the ultrafiltration the final retentate was lyophilized. Thecrude product was purified by preparative HPLC (C18 YMC-ODS AQ, 10 μm,acetonitrile in water+0.1% formic acid, 1% to 10%) to yield 14.5 mg ofthe title compound.

UPLC (ACN-HCOOH): Rt.=0.34 min.

MS (ES⁺): m/z (z=2)=1272.9 (M+2H)²⁺

Example 10 Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxylato-methyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[a[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]propyl)-amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate

Example 10a

Tert-butyl{4,10-bis(2-tert-butoxy-2-oxoethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]-propyl}amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate

6.6 mg (49.8 μmol, 1 eq.) 2,2-Bis(aminomethyl)propane-1,3-diamine (seeW. Hayes et al., Tetrahedron 59 (2003), 7983-7996) were dissolved in 7mL DMSO. After adding of 77 mg (0.6 mmol, 12 eq.),N,N-diisopropylethylamine and 400 mg (0.6 mmol, 12 eq.) tri-tert-butyl2,2′,2″-(10-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (see M. Galibert et al., Bioorg. Med.Chem. Letters 2010 (20), 5422-5425 and J. Am. Chem. Soc. 2006, 128, p.15072-15073; S3-5) the resulting reaction mixture was stirred and heatedover night at 50° C. The cooled solution was concentrated under reducedpressure. The crude product was used without further characterizationfor the next chemical step.

Example 10b{4,10-bis(carboxymethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-cyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(carboxymethyl)-1,4,7,10-tetra-azacyclododecan-1-yl]acetyl}amino)methyl]propyl}amino)ethyl]-1,4,7,10-tetraazacyclo-dodecan-1-yl}aceticacid

The crude tert-butyl{4,10-bis(2-tert-butoxy-2-oxoethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]-propyl}amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetatefrom example 10a was dissolved in 40 mL TFA. The resulting solution wasstirred overnight at room temperature and was concentrated under reducedpressure. The crude product was used without further characterizationfor the next chemical step.

Example 10 Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxylato-methyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]propyl}-amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate

The crude{4,10-bis(carboxymethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]propyl}amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}aceticacid from example 10b was dissolved in 10 mL water. After addition of326 mg of tris(acetato-kappaO)gadolinium tetrahydrate the pH value ofthe resulting solution was adjusted to 3.5-4.5 by addition of aqueoussodium hydroxide solution. The reaction mixture was heated understirring overnight at 70° C. The resulting solution was ultrafilteredwith water using a 1 kDa membrane and the final retentate waslyophilized. The crude product was purified by RP-chromatographyyielding 65 mg (28 μmol, 46%) of the title compound.

UPLC (ACN-HCOOH): Rt.=0.40 min.

MS (ES⁺): m/z (z=2)=1149.7 (M+2H)²⁺, m/z (z=3)=766.0 (M+3H)³⁺.

Example 11 Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}-methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

Example 11a Tert-butyl[4,10-bis(2-tert-butoxy-2-oxoethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}-methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

2.99 g (4.75 mmol, 12eq.)N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycine(see M. Suchy et al., Org. Biomol. Chem. 2010, 8, 2560-2566) and 732 mg(5.70 mmol, 14.4 eq.) ethyldiisopropylamine were dissolved in 40 mLN,N-dimethylformamide. After addition of 2.17 g1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU; 5.70 mmol, 14.4 eq.) the reactionmixture was stirred for 15 minutes at room temperature. 100.1 mg (396μmol, 1 eq.) 2,2-bis(ammoniomethyl)propane-1,3-diaminium tetrachloride(see W. Hayes et al., Tetrahedron 59 (2003), 7983-7996) and 982.7 mg(7.60 mmol, 19.2 eq.) ethyldiisopropylamine were added and the resultingreaction mixture was stirred over night at 50° C. The cooled solutionwas concentrated under reduced pressure. The crude product was usedwithout further characterization for the next chemical step.

Example 11 b[4,10-bis(carboxymethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-cyclododecan-1-yl]acetyl}amino)acetyl]amino}methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid

The crude tert-butyl[4,10-bis(2-tert-butoxy-2-oxoethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}-methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetatefrom example 11 a was dissolved in 125 mL TFA. The resulting solutionwas stirred for 2 hours at 70° C., then overnight at room temperature,and was concentrated under reduced pressure.

The oily product was dissolved in 200 mL water, was isolated bylyophilisation, and was used without further characterization for thenext chemical step.

Example 11 Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}-methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate

The crude[4,10-bis(carboxymethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]aceticacid from example 11 b was dissolved in 100 mL water. After addition of2.89 g of tris(acetato-kappaO)gadolinium tetrahydrate the pH value ofthe resulting solution was adjusted to 3.0-3.5 by addition of aqueoussodium hydroxide solution. The reaction mixture was heated understirring for 24 hours at 70° C. The resulting solution was ultrafilteredwith water using a 1 kDa membrane and the final retentate waslyophilized. The crude product was purified by RP-chromatographyyielding 296 mg (120 μmol, 30%) of the title compound.

UPLC (ACN-HCOOH): Rt.=0.41 min.

MS (ES⁺): m/z (z=2)=1262.8 (M+2H)²⁺, m/z (z=3)=841.5 (M+3H)³⁺.

Reference Compound 1

Gadovist® (gadobutrol, Bayer AG, Leverkusen, Germany)

Reference Compound 2

Magnevist® (gadopentetate dimeglumine, Bayer AG, Leverkusen, Germany)

Reference Compound 3

Primovist® (gadoxetate disodium, Bayer AG, Leverkusen, Germany)

Reference Compound 4

Gadomer-17 was synthesized as described in EP0836485B1, Example 1 k.

In Vitro and In Vivo Characterization of Example Compounds

Examples were tested in selected assays one or more times. When testedmore than once, data are reported as either average values or as medianvalues, wherein

-   -   the average value, also referred to as the arithmetic mean        value, represents the sum of the values obtained divided by the        number of times tested, and    -   the median value represents the middle number of the group of        values when ranked in ascending or descending order. If the        number of values in the data set is odd, the median is the        middle value. If the number of values in the data set is even,        the median is the arithmetic mean of the two middle values.

Examples were synthesized one or more times. When synthesized more thanonce, data from assays represent average values or median valuescalculated utilizing data sets obtained from testing of one or moresynthetic batch.

Example A Relaxivity Measurements at 1.4 T

Relaxivity measurements at 1.41 T were performed using a MiniSpec mq60spectrometer (Bruker Analytik, Karlsruhe, Germany) operating at aresonance frequency of 60 MHz and a temperature of 37° C. The T₁relaxation times were determined using the standard inversion recovery(IR) method with a fixed relaxation delay of at least 5×T₁. The variableinversion time (TI) was calculated automatically by the standardsoftware of the MiniSpec mq60 (8 steps). The T₂ measurements were doneby using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence, applying arelaxation delay of at least 5×T₁.

Each relaxivity measurement was performed using three different Gdconcentrations (3 concentrations between 0.05 and 2 mM). The T₁ and T₂relaxation times of the example compounds 1-10 were measured indifferent media for example in water, fetal bovine serum (FBS, Sigma,F7524) and human plasma.

Human plasma preparation: For each experiment fresh blood was taken froma volunteer using 10 mL citrate-tubes (Sarstedt S-Monovette 02.1067.001,10 mL, Citrate). The 10 mL citrate-tubes were carefully inverted 10times to mix blood and anticoagulant and centrifuged for 15 minutes at1811 g at room temperature (Eppendorf, Centrifuge 5810R).

The relaxivities r_(i) (where i=1, 2) were calculated on the basis ofthe measured relaxation rates R_(i) in water and plasma:

R _(i) =R _(i(0)) +r _(i)[C _(Gd)],

where R_(i(0)) represent the relaxation rate of the respective solventand C_(Gd) the concentration of the compound normalized to theGadolinium. The Gadolinium concentrations of the investigated solutionswere verified by Inductively Coupled Plasma Mass Spectroscopy (ICP-MSAgilent 7500a, Waldbronn, Germany). The determined relaxivity values aresummarized in Table 1.

TABLE 1 Relaxivities of investigated compounds in water, fetal bovineserum (FBS) and human plasma at 1.41 T and relaxivities of Referencecompounds 1-4 (RC1-RC4) at 1.5 T in water and bovine plasma. All valueswere measured at 37° C., are normalized to Gd and given in L mmol⁻¹ s⁻¹.Example r₁ r₂ r₁ r₂ r₁ human r₂ human No water* water* FBS* FBS* plasma*plasma* 1 11.1 12.9 13.2 16.3 13.0 19.5 2 12.1 14.2 13.4 16.4 13.9 17.63 10.1 11.7 11.5 13.7 11.8 14.7 3-1 9.5 11.1 n.d. n.d. 10.4 13.1 3-2 9.410.8 n.d. n.d. 11.4 14.2 4 11.5 13.5 13.3 16.0 13.2 16.5 5 13.0 15.214.6 18.1 14.3 17.7 6 13.4 15.7 14.2 17.5 14.6 18.6 7 10.8 12.6 11.714.4 12.1 14.9 8 12.5 14.5 14.5 17.9 14.6 18.1 9 7.4 8.5 8.8 10.4 n.d.n.d. 10  7.3 8.3 9.2 10.7 9.7 11.3 RC1{circumflex over ( )} 3.3 3.9 5.26.1 n.d. n.d. RC2{circumflex over ( )} 3.3 3.9 4.1 4.6 n.d. n.d.RC3{circumflex over ( )} 4.7 5.1 6.9 8.7 n.d. n.d. RC4{circumflex over( )} 17.3 22 16 19 n.d. n.d. *values are depicted in L mmo1⁻¹ s⁻¹{circumflex over ( )}Relaxivities from reference compounds from Rohreret. al. (Invest. Radiol. 2005; 40, 11: 715-724), bovine plasma (KreaberGmbH, Pharmaceutical Raw Material, Ellerbek, Germany)

Relaxivity measurements at 3.0 T were performed with a whole body 3.0 TMRI Scanner (Philips Intera, Philips Healthcare, Hamburg, Germany) usinga knee-coil (SENSE-Knee-8, Philips Healthcare, Hamburg, Germany). Thesample tubes (CryoTubetm Vials, Thermo Scientific 1.8 mL, Roskilde,Denmark) were positioned in 3 rows of 4 and 5 tubes in a plastic holderin a box filled with water. The temperature was adjusted to 37° C. Forthe MRI sequence, the shortest possible echo-time (TE) with 7.46milliseconds was used. The inversion times were chosen to optimize thesequence to measure T₁ values corresponding to the estimated T₁ range ofall relaxation times of contrast media containing solutions. Thefollowing inversion times (TIs) were applied: 50, 100, 150, 200, 300,500, 700, 1000, 1400, 2100, 3200, and 4500 milliseconds. The sequencewas run with a constant relaxation delay of 3.4 seconds after theregistration of the last echo (variable TR in the range from 3450 to7900 milliseconds). For details of the fit procedure, see Rohrer et. al.(Invest. Radiol. 2005; 40, 11: 715-724). The experimental matrix of thephantom measurement was 320×320.

The relaxivities were evaluated using three different concentrations ofeach compound (3 concentrations between 0.05 and 2 mM).

The T₁ relaxation times of the Example compounds 1-6 were measured inwater and human plasma. Human plasma preparation: For each experimentfresh blood was taken from a volunteer using 10 mL citrate-tubes(Sarstedt S-Monovette 02.1067.001, 10 mL, Citrate). The 10 mLcitrate-tubes were carefully inverted 10 times to mix blood andanticoagulant and centrifuged for 15 minutes at 1811 g at roomtemperature (Eppendorf, Centrifuge 5810R).

The relaxivities r_(i) (where i=1, 2) were calculated on the basis ofthe measured relaxation rates R_(i) in water and plasma:

R _(i) =R _(i(0)) +r _(i)[C _(Gd)],

where R_(i(0)) represent the relaxation rate of the respective solventand C_(Gd) the concentration of the compound normalized to theGadolinium (Table 2).

TABLE 2 Relaxivities (normalized to Gd) in water and human plasma at 3.0T and 37° C. [L mmol⁻¹ s⁻¹] Example No r₁ water* r₁ human plasma* 1 9.5± 0.2 10.8 ± 0.1 2 9.2 ± 0.3 11.4 ± 0.1 3 9.2 ± 0.3 10.2 ± 0.2 3-1 8.9 ±0.2 10.1 ± 0.1 3-2 9.0 ± 0.4 11.4 ± 0.2 4 10.1 ± 0.2  11.8 ± 0.3 5 10.8± 0.3  12.4 ± 0.2 6 11.3 ± 0.4  12.8 ± 0.3 RC1{circumflex over ( )} 3.2± 0.3  5.0 ± 0.3 RC2{circumflex over ( )} 3.1 ± 0.3  3.7 ± 0.2RC3{circumflex over ( )} 4.3 ± 0.3  6.2 ± 0.3 RC4{circumflex over ( )}13.0 ± 0.7  13 ± 1 *Average ± standard deviation, values are depicted inL mmol⁻¹ s⁻¹

Example B Pharmacokinetic Parameters

Pharmacokinetic parameters of the compound of Example 3 were determinedin male rats (Han-Wistar, 220-230 g, n=3). The compound was administeredas a sterile aqueous solution (52.5 mmol Gd/L) as a bolus in the tailvein of the animals. The dose was 0.1 mmol Gd/kg. Blood was sampled 1,3, 5, 10, 15, 30, 60, 90, 120, 240, 360, 480 and 1440 min post injectionand the Gd concentration was determined by Inductively Coupled PlasmaMass Spectroscopy (ICP-MS Agilent 7500a, Waldbronn, Germany). The bloodlevel was converted to plasma concentrations by division by 0.625(plasma fraction of rat blood, assuming strictly extracellulardistribution). As a control, 3 animals were treated in the same way withGadovist®, a low molecular weight contrast agent. The time courses ofthe blood plasma levels are shown in FIG. 1.

The fit of the obtained data to a three compartment model(Phoenix—WinNonlin) yielded the pharmacokinetic parameters which areshown in Table 3.

TABLE 3 Time courses of blood plasma levels Gadoviste ® Example 3Parameter unit mean SD mean SD t½ α Half-life, compartment V1 [min] 1.60.4 1.7 0.3 t½ β Half-life, compartment V2 [min] 20.5 1.9 18.2 3.4 t½ ΥHalf-life, compartment V3 [min] 232 126 133 22.0 MRT Mean residence time[min] 30.1 3.8 24.1 4.4 AUC∞ Area under the curve (to [μmol/l * min]11500 1180 9040 1220 infinity) V_(c) (V1) Volume, central compartment[l/kg] 0.14 0.01 0.11 0.01 V1 V2 Volume, compartment V2 [l/kg] 0.12 0.010.15 0.01 V1 + V2 Volume, compartments [l/kg] 0.25 0.02 0.26 0.01 V1 +V2 V_(d,ss) Volume of distribution at [l/kg] 0.28 0.02 0.28 0.01 steadystate Cl_(tot) Total Clearance [ml/min * kg] 9.30 0.9 11.8 1.7

Example C

Excretion and Residual Organ Gadolinium Concentration after 5 Days

The excretion and organ distribution of Example 3 were determined inmale rats (Han-Wistar, 100-110 g, n=3). The compound was administered asa sterile aqueous solution (54 mmol Gd/L) as a bolus in the tail vein ofthe animals. The dose was 0.1 mmol Gd/kg. Urine was collected in thefollowing time periods 0-1 h, 1-3 h, 3-6 h, 6-24 h, 1-2 d and 2-5 d postinjection and feces 0-1 d, 1-2 d and 2-5 d post injection. As a control,3 animals were treated in the same way with Gadovist®, a low molecularweight contrast agent. On day 7 the animals were sacrificed and thefollowing organs were excised: blood, liver, kidney, spleen, heart,lung, brain, mesenteric lymph nodes, muscle, skin, stomach, gut, boneand bone marrow. The remaining carcass was freeze dried and ground to afine powder. The Gd concentration in the organs and the carcass wasdetermined by ICP-MS (ICP-MS Agilent 7500a, Waldbronn, Germany). Theresults of the organ distribution of Example 3 and Reference compound 1(Gadovist®) are summarized in Table 4. The Example 3 is excreted quicklyvia the kidneys. After 3 h 95.8%±3.4% of the injected dose was found inurine and 96.9%±3.7% after 5 days. About 1.4%±0.6% was excreted via thefeces. Less than 0.5% of the administered dose was present in the body 7days after the injection. The individual organs contained less than0.03% of the injected dose, except the kidney which is the excretionorgan.

TABLE 4 Excretion and organ distribution of Gadovist ® and Example 3 inrats Gadoviste ® [% Dose] Example 3 [% Dose] Time period Urine Urinepost injection 0-1 h 91.28 ± 2.69%  90.36 ± 4.4%   1-3 h 7.38 ± 1.50%5.43 ± 1.04% 3-6 h 0.22 ± 0.08% 0.46 ± 0.38% 6-24 h 0.28 ± 0.03% 0.17 ±0.02% 1-2 d 0.20 ± 0.02% 0.14 ± 0.01% 2-5 d 0.64 ± 0.18% 0.34 ± 0.03%Time period Feces Feces post injection 0-1 d 1.47 ± 1.38% 1.13 ± 0.62%1-2 d 0.13 ± 0.08% 0.10 ± 0.02% 2-5 d 0.13 ± 0.02% 0.13 ± 0.01% Timepoint Σ organs and carcass Σ organs and carcass post injection 7 d  0.50± 0.07% 0.49 ± 0.01% Total recovery 101.9 ± 0.4%  98.8 ± 3.1% 

Example D Chemical Stability

Examples 1, 2, 3 and 6 were separately dissolved in 10 mM Tris-HClbuffer, pH 7.4 at a final concentration of 5 mmol Gd/L. An aliquot wasremoved and the rest of the clear and colorless solution was autoclavedat 121° C. for 20 m in. After autoclaving, the solution was still clearand colorless. The aliquot removed before and after autoclaving wasanalyzed by HPLC-ICP-MS to determine the integrity of the compound.

HPLC: Column: Hypercarb 2.5 mm×15 cm. Solvent A: 0.1% formic acid inwater. Solvent B: acetonitrile. Gradient from 100% A to 5% A+95% B in 10min. Flow 1 ml/min. Detection by ICP-MS, tuned to ¹⁵⁸Gd. Thechromatograms, displaying the intensity of the detected Gd, werevisually compared. No changes in the chromatograms before and afterautoclaving were detected. The compounds were stable during theautoclaving procedure.

Example E

Gadolinium Release after the Addition of Zinc and Phosphate

The proton relaxometric protocol for the transmetallation assessment forthe stability determination of MRI contrast media is described inLaurent S., et al. (Invest. Radiol. 2001; 36, 2: 115-122). The techniqueis based on measurement of the evolution of the water protonparamagnetic longitudinal relaxation rate in phosphate buffer (pH 7.00,26 mmol/L, KH₂PO₄ Merck, Hessen, Germany) containing 2.5 mmol/Lgadolinium complex and 2.5 mmol/L ZnCl₂ Sigma-Aldrich, Munich, Germany).One hundred microliters of a 250 mmol/L solution of ZnCl₂ were added to10 mL of a buffered solution of paramagnetic complex (Referencecompounds 1-4 and Example 3). The mixture was vigorously stirred, and300 μL were taken out for the relaxometric study at 0 min, 60 min, 120min, 3 h, 4 h, 5 h, 24 h, 48 h and 72 h. The measurements were performedon a MiniSpec mq60 spectrometer (Bruker Analytik, Karlsruhe, Germany) at60 MHz and 37° C. The results of Example 3 in comparison to Referencecompound 1 (Gadovist®), Reference compound 2 (Magnevist®) and Referencecompound 3 (Primovist®) are shown in FIG. 2. If Gadoliniumtransmetallation is triggered by the Zn²⁺ ions in a phosphate-bufferedsolution, then free released Gd³⁺ would react with the free PO₄ ³⁻ ionsto form GdPO₄. Due to the low solubility of GdPO₄, a part of theGadolinium precipitates as solid and has no further influence on thelongitudinal relaxation rate of water. A decrease of the protonrelaxation rate would be observed for Gadolinium chelates with a lowstability [see linear contrast media in FIG. 2: Reference compounds 2(Magnevist®) and 3 (Primovist®)]. The stability of Example 3 iscomparable to the high stability of Reference compound 1 (Gadovist®).

Example F Gd-Complex Stabilities in Human Plasma at 37° C., 15 d

Examples 3 and 10 were separately dissolved in human plasma at 1 mmolGd/L. As a reference for released Gd³+, 0.1 mmol/L Gadolinium chloride(GdCl₃) was dissolved in human plasma. The plasma samples were incubatedfor 15 days at 37° C. under 5% CO 2 atmosphere to maintain the pH at7.4. Aliquots were taken at the start and end of the incubation. Theamount of Gd³⁺ released from the complexes was determined byHPLC-ICP-MS. Column: Chelating Sepharose (HiTrap, 1 mL). Solvent A: 10mM BisTris-HCl pH 6.0. Solvent B: 15 mM HNO₃. Gradient: 3 min at 100% A,from 3 to 10 min at 100% B. Flow 1 mL/min. Detection by ICP-MS, tuned to¹⁵⁸Gd. The chromatograms, displaying the intensity of the detected Gd,were evaluated by peak area analysis. The size of the peak of Gd³⁺,eluting after the change from solvent A to B, was recorded. For bothcompounds the increase of this peak and thus the release of Gd³⁺ werebelow the limit of quantification (<0.1% of the injected total amount ofGadolinium). Both Gd-complexes are stable under physiologicalconditions.

Example G Water Solubility

The water solubility of the compounds was determined at room temperature(20° C.) in 0.5 mL buffer solution (10 mM Tris-HCl) in themicrocentrifuge tubes (Eppendorf, 2.0 mL safe-lock caps). The solidcompound was added stepwise to the buffer solution. The suspension wasmixed using a shaker (Heidolph Reax 2000) and treated 5 min in anultrasound bath (Bandelin, Sonorex Super RK255H) The suspension wasstored at room temperature (20° C.) over night and final Gadoliniumconcentration was determined by inductively coupled plasma massspectrometry (ICP-MS). The results are summarized in Table 5.

TABLE 5 Solubilities of compounds in water at 20° C. Example SolubilityNo [mmol Gd/L] 1 >1200 2 >1200 3 >1400 4 >1200 5 >1100 6 >1100 7 >14008 >1000 9  >800 10   >800

Example H Contrast-Enhanced Magnetic Resonance Angiography (CE-MRA)

The potential of a significant dose reduction was shown by anintraindividual comparison of 100 μmol Gadolinium per kilogram bodyweight [100 μmol Gd/kg bw], which is comparable to the human standarddose, and a low dose protocol using 30 μmol Gadolinium per kilogram bodyweight. Reference compound 1 (Gadovist®), as an approved representativeof the Gadolinium-based MRI contrast agents, was used in both doseprotocols (100 μmol Gd/kg bw and 30 μmol Gd/kg bw) and compared toExample 3 (30 μmol Gd/kg bw).

The contrast-enhanced magnetic resonance angiography study was performedat a clinical 1.5 T Scanner (Magnetom Avanto, Siemens Healthcare,Erlangen, Germany). For optimal signal exploitation, a standard spinecoil was used for the data acquisition. The study was done using maleNew Zealand white rabbits (weight 2.5-2.9 kg, n=6, Charles RiverKisslegg). All animals were initially anesthetized using a bodyweight-adjusted intramuscular injection of a mixture (1+2) of xylazinehydrochloride (20 mg/mL, Rompun 2%, Bayer Vital GmbH, Leverkusen,Germany) and ketamine hydrochloride (100 mg/mL, Ketavet, Pfizer,Pharmacia GmbH, Berlin, Germany) using 1 mL/kg body weight. Thecontinuous anesthesia of the intubated animals (endotracheal tube,Rueschelit Super Safe Clear, cuff 3.0 mm, Willy Ruesch AG, Kernen,Germany) was achieved by the intravenous injection of 0.9 mg propofolper kilogram per hour (10 mg/mL, Propofol-Lipuro 1%, B. Braun MelsungenAG, Melsungen, Germany). The continuous intravenous injection wasperformed using a MR infusion system (Continuum MR Infusion System,Medrad Europe B. V., A E Beek, Germany). The tracheal respiration (SV900C, Maquet, Rastatt, Germany) was performed with 55% oxygen, fortybreaths per minute and a breathing volume of 7 mL per kilogram bodyweight per minute.

Based on a localizer sequence oriented in coronal, axial, and sagittaldirections, the anatomic course of the aorta was acquired. The time topeak was determined using a small intravenous test bolus (0.25mL/2.5-2.7 kg or 0.3 mL/2.8-2.9 kg bw, Reference compound 1) and a 3DFLASH sequence (test bolus sequence: repetition time: 36.4 millisecond,echo time 1.45 millisecond, flip angle: 30 degree, spatial resolution:1.0×0.8×17 mm). The angiography 3D FLASH sequence was characterized by arepetition time of 3.24 milliseconds, an echo time of 1.17 milliseconds,a flip angle of 25 degree and a slice thickness of 0.94 mm. The field ofview of 141×300 mm was combined with a matrix of 150×320 resulting in aspatial resolution of 0.9×0.9×0.9 mm and a whole acquisition time of 13seconds per 3D block. The 3D FLASH sequence was performed once beforeand immediately after injection of the contrast agent. The time intervalfor the intraindividual comparison between the different contrast agentapplications was twenty to thirty minutes (n=3 animals).

The resulting magnetic resonance angiographs of the intraindividualcomparison in rabbits are depicted in FIG. 3A-3C: FIG. 3A shows 30 μmolGd/kg bw Reference compound 1 (Gadovist®); FIG. 3B shows 30 μmol Gd/kgbw Example 3 and FIG. 3C shows 100 μmol Gd/kg bw Reference compound 1.The contrast enhancement of the low dose protocol with Example 3 (FIG.3B) is comparable to that of the standard dose of Reference compound 1(FIG. 3C). Furthermore, the image quality of the low dose protocol ofExample 3 (FIG. 3B) is significantly better than the low dose protocolof Reference compound 1 (FIG. 3A). The angiography study demonstratesthe potential of Example 3 for a significant dose reduction.

Example J Whole Body Imaging

Classical extracellular Gadolinium-based contrast agents exhibit a rapidextracellular passive distribution in the whole body and are excretedexclusively via the kidney. The fast extracellular distribution in thewhole body enables the classical imaging possibilities as for exampleangiography and the imaging of the central nervous system, extremities,heart, head/face/neck, abdomen and breast. The comparability of thepharmacokinetic and diagnostic behavior of Reference compound 1(Gadovist®) and other ECCM has been shown and forms the basis forbridging the efficacy to all body parts usually imaged in the diagnosticworkup of a variety of diseases (Tombach B., et. al., Eur. Radiol. 2002;12(6):1550-1556). The described contrast-enhanced magnetic resonancestudy compares the pharmacokinetic distribution and the diagnosticperformance of Example 3 to Reference compound 1 (Gadovist®), as anapproved representative of the Gadolinium-based MRI contrast agents.

To demonstrate that Example 3 has the same mode of action, MRI signalintensity over time and Gd concentrations were determined in varioustissues. The study was performed with a clinical whole body MRI equippedwith body spine coil, abdomen flex coil, neck coil (1.5 T MagnetomAvanto, Siemens Healthcare, Erlangen, Germany). The study was done usingmale New Zealand white rabbits (weight 2.3-3.0 kg, n=8, Charles RiverKisslegg). All animals were initially anesthetized using a bodyweight-adjusted intramuscular injection of a mixture (1+2) of xylazinehydrochloride (20 mg/mL, Rompun 2%, Bayer Vital GmbH, Leverkusen,Germany) and ketamine hydrochloride (100 mg/mL, Ketavet, Pfizer,Pharmacia GmbH, Berlin, Germany) using 1 mL/kg body weight. Thecontinuous anesthesia of the intubated animals (endotracheal tube,Rueschelit Super Safe Clear, cuff 3.0 mm, Willy Ruesch AG, Kernen,Germany) was achieved by the intravenous injection of 0.9 mg propofolper kilogram per hour (10 mg/mL, Propofol-Lipuro 1%, B. Braun MelsungenAG, Melsungen, Germany). The continuous intravenous injection wasperformed using a MR infusion system (Continuum MR Infusion System,Medrad Europe B. V., A E Beek, Germany). The tracheal respiration (SV900C, Maquet, Rastatt, Germany) was performed with 55% oxygen, fortybreaths per minute and a breathing volume of 7 mL per kilogram bodyweight per minute.

Dynamic MRI measurements up to 22 min post injection with subsequentquantitative signal analysis (Siemens Mean Curve software (SYNGO TaskCard, Siemens Healthcare, Erlangen, Germany), were performed for threedifferent regions head and neck (brain, tongue, chops muscle, neckmuscle), abdomen (spleen, liver, blood) and pelvis (extremity muscle).For the three different slice groups a 3D T1-weighted Vibe sequence wasused (TR=4.74 ms, TE=2.38, flip=10°, 1:29 min). The dynamic measurementsof the three slice groups (Head/Neck: 1:29 min, Abdomen: 0:49 min,Pelvis: 1:16 min) were done up to 22 min post injection: 1. Head/Neck:baseline, 1.4, 5.2, 8.9, 12.8, 16.5, 20.4 min, 2. Abdomen: baseline,0.5, 4.3, 8.1, 11.9, 15.7, 19.5 min and 3. Pelvis: baseline, 2.9, 6.7,10.5, 14.4, 18.1, 22.0 min. At 30 min post injection the animals weresacrificed and the Gd concentrations were measured using InductivelyCoupled Plasma Mass Spectroscopy (ICP-MS Agilent 7500a, Waldbronn,Germany) in the following tissue samples: blood, brain, tongue, liverand extremity muscle. A quantitative image evaluation was performed forthe 30 min time point p.i. due to the combination of the quantitativeICP-MS Gadolinium concentrations and the MRI region-of-interestanalysis.

The administration of the contrast agent leads to a signal increase inthe vascular system and in the extravascular, extracellular space of thebody. The signal enhancement is based on the pharmacokinetic andphysicochemical properties of the contrast agents. FIGS. 4A and 4B showrepresentative images of the head and neck region before and 1.4 minafter administration of Example 3 (FIG. 4A) and Reference compound 1(FIG. 4B). FIGS. 5A and 5B show representative abdominal images beforeand 0.5 min after administration of Example 3 (FIG. 5A) and Referencecompound 1 (FIG. 5B). FIGS. 6A and 6B show representative images of thepelvis region before and 0.5 min after administration of Example 3 (FIG.6A) and Reference compound 1 (FIG. 6B). All images show a clear signalenhancement for example in the heart, tongue, aorta, kidney, liver,spleen, the whole vascular system and muscles.

The signal-time curves show the signal change over time after contrastagent administration and represent the contrast agent pharmacokineticsin the respective tissue (FIG. 7). In all investigated tissues a rapidincrease of signal intensity was observed after contrast agent injectionwhich was followed by a continuous signal decrease. The degree of thesecontrast enhancements is tissue specific. However, no differences in thetime course of contrast enhancements were observed between Example 3(dotted line) and Reference compound 1 (solid line). This demonstratesidentical pharmacokinetic properties and shows that Example 3 issuitable for different body regions (FIG. 7). The amplitude of contrastenhancement depends on tissue characteristics, especially on tissueperfusion and the physicochemical properties, especially on relaxivity.As expected from the approximately 2-fold higher relaxivity (see ExampleA) the contrast enhancement using Example 3 is higher compared to thatof Reference compound 1.

The relation between Gadolinium concentration and MRI signal change wereinvestigated by comparing the amount of Gadolinium in tissue 30 min p.i.with the signal change at the MRI measurement performed at 19.5 min p.i.(abdomen), 20.4 min p.i. (head and neck) and 22.0 min p.i. (pelvis). Therespective data for Example 3 and Reference compound 1 are shown inFIGS. 8A and 8B, respectively. A linear correlation between theGadolinium concentrations in various tissues and the respective MRIsignal changes were observed. This demonstrates that the efficacy ofExample 3 (FIG. 8A) and Reference compound 1 (FIG. 8B) are independentof the body region or tissue investigated. A slight deviation from thiscorrelation was observed for the spleen, which shows a higher MRI signalenhancement than it would be expected from the Gadolinium tissueconcentration. This was observed for both contrast agents and relates tothe significantly higher blood volume of the spleen in comparison toother organs and tissues. Consequently the spleen loses much of itsGadolinium concentration by the exsanguination which in turn results ina mismatch between in-vivo imaging and ex-vivo Gadolinium determination.The correlation between signal change and tissue Gadoliniumconcentration of all other tissues and organs, which represents therespective relaxivity, depends on the efficacy of the contrast agentused. A larger slope was determined for Example 3 (1.9) (FIG. 8A) thanfor Reference compound 1 (1.0) (FIG. 8B), which is in good agreementwith the known higher relaxivity of Example 3 (FIGS. 8A and 8B; see alsorelaxivity data described in Example A).

Example K Dynamic CT Diffusion Phantom Study

As indicated in Example A, the Reference compound 4 has a relaxivitywhich is in a similar range as the compounds of the present invention.Following intravenous injection, all clinically approved small monomerGBCAs (gadopentetate dimeglumine, gadoterate meglumine, gadoteridol,gadodiamide, gadobutrol and gadoversetamide) distribute in the blood andextravascular/extracellular space by passive distribution (Aime S., et.al., J. Magn. Reson. Imaging 2009; 30, 1259-1267). Contrast agents witha high protein binding, for example gadofosveset trisodium with aprolonged period in the blood vessels caused by the reversible bindingto HSA, or large hydrodynamic sizes as for example Reference compound 4are hindered to pass the vessel wall. For good imaging results a fastdiffusion through the vessel walls is required due to the fast renalexcretion of GBCAs.

The described dynamic CT diffusion study compares the ability ofExamples 1, 2, 3, 4, 5, 6 and Reference compounds 1 and 4 to pass asemipermeable membrane (20 kDa). A 128-row clinical CT device (SOMATOMDefinition, 128; Siemens Healthcare, Forchheim, Germany) was used tomonitor the diffusion through a semipermeable membrane at 100 kV and 104mA. Single measurements were performed at 0 min, 1 min, 2 min, 3 min, 5min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 2 h, 3 h, 5 h, 7 h,22 h, 24 h, 30 h, 46 h and 48 h after placing the dialysis cassette(Slide-A-Lyser, 20,000 MWCO, 0.1-0.5 mL Capacity, Thermo Scientific,Roskilde, Denmark) filled with contrast agent in fetal bovine serumsolution (FBS, Sigma, F7524). The images were reconstructed with a slicethickness of 2.4 mm and a B30 convolution kernel. The used concentrationin the dialysis cassettes of the investigated Examples 1, 2, 3, 4, 5, 6and Reference compounds 1 and 4 was 20 mmol Gd/L.

The imaging results for all investigated Examples and the Referencecompounds 1 and 4 for the time points 0 min and 48 h after placing thecassettes in the FBS solution are depicted in FIGS. 9A and 9B,respectively. For image analysis, regions of interest were manuallydrawn on 1 centrally located slice for each time point (a representativemeasurement region is indicated by the arrow in FIG. 9A: Image 1A). Theresults of the Hounsfield units (HU) of the analyzed regions over timeare shown in FIG. 10. The calculated diffusion half-lives of theinvestigated Examples and Reference compounds are summarized in Table 6.

TABLE 6 Diffusion half-live through a semipermeable membrane (20 kDa)Example Diffusion half-life (20 kDa) No [h] 1 39 2 39 3 11 4 21 5 24 636 RC 1  2 RC 4 ~90000The FIG. 10 and the calculated half-life data show, similar to theReference compound 1 (Gadovist®) and in contrast to the Referencecompound 4, that the Examples 1-6 are able to pass the semipermeablemembrane. Furthermore, the data of the investigated compounds showcontrary to other high relaxivity agents, which have a high proteinbinding or very slow tumbling rates (e.g. Reference compound 4), thatthe compounds of the present invention have hydrodynamic dimensionswhich can overcome barriers in a timely manner. These findings indicatethe ability of the compounds of the invention to overcome barriers asfor example endothelial walls in the vascular system, which is arequirement for whole body imaging.

Example L Evaluation of Potential Side Effects

None of the investigated example compounds showed undesired negativeside effects in animals after application. Additionally the off targetactivity of the Example 3 was screened in commercial radioligand bindingand enzyme assays (LeadProfilingScreen®, Eurof ins Panlabs, Taipei,Taiwan) and revealed no critical finding.

Example M

Contrast-enhanced MRI of brain tumors in rats

The potential of a significant dose reduction was shown by anintraindividual comparison of 0.3 mmol Gadolinium per kilogram bodyweight (300 μmol Gd/kg bw) and a low dose protocol using 0.1 mmolGadolinium per kilogram body weight (100 μmol Gd/kg bw). Referencecompound 1 (Gadovist®), as an approved representative of theGadolinium-based MRI contrast agents, was used in both dose protocols(0.3 mmol Gd/kg bw and 0.1 mmol Gd/kg bw) and compared to Example 3 (0.1mmol Gd/kg bw).

GS9L cell line (European Collection of Cell Cultures, Cancer Res. 1990;50:138-141; J. Neurosurg. 1971; 34:335) were grown in Dulbecco'sModified Eagle Medium (DMEM, GlutaMAX™, Ref: 31966-021, Gibco)supplement with 10% fetal bovine serum (FBS, Sigma F75249) and 1%Penicillin-Streptomycin (10.000 units/mL, Gibco). The study was doneusing male Fisher rats (F344, weight 170-240 g, n=4, Charles RiverKisslegg). Inoculation was performed under ketamine/xylazine anesthesiausing a body weight-adjusted intramuscular injection of a mixture (1+2)of xylazine hydrochloride (20 mg/mL, Rompun 2%, Bayer Vital GmbH,Leverkusen, Germany) and ketamine hydrochloride (100 mg/mL, Ketavet,Pfizer, Pharmacia GmbH, Berlin, Germany) using 1 mL/kg body weight. Fororthotopically intracerebral implantation anesthetized animals werefixed in a stereotactic apparatus and 1.0E+06 GS9L cells suspended in avolume of 5 μl medium were injected slowly into the brain using aHamilton syringe.

The contrast-enhanced MRI study was performed at a clinical 1.5 TScanner (Magnetom Avanto, Siemens Healthcare, Erlangen, Germany). A rathead coil (coil and animal holder for rats, RAPID Biomedical GmbH) wasused for the data acquisition. The rats were anesthetized using amixture of isoflurane (2.25%), oxygen gas (ca. 0.5 L/min) and nitrousoxide (flow ca. 1 L/min). MR Imaging was done using a 3D turbo-spin echosequence (12 1 mm slices in a 3 D block, field of view: 80 mm (33%oversampling), repetition time: 500 millisecond, echo time 19millisecond, spatial resolution: 0.3×0.3×1.0 mm). The animals wereimaged at two consecutive days. The first day the Reference compound 1(Gadovist®) and the Example 3 were intraindividually compared at thesame dose of 0.1 mmol Gd/kg bw, which is comparable to the humanstandard dose. The second day the Reference compound 1 (Gadovist®) at0.3 mmol Gd/kg bw, which is comparable to the triple human dose(clinically approved in certain CNS indications), was compared to thestandard dose of Example 3 (0.1 mmol Gd/kg bw). The resulting MR imagesof the GS9L rat brain tumors are depicted in FIGS. 11A and 11B: FIG. 11Ashows an intraindividual comparison of Reference compound 1 (Gadovisi®)and Example 3 at the same dose of 0.1 mmol Gd/kg body weight (bw).Example 3 showed at the same dose higher lesion-to-brain contrast and anexcellent demarcation of the tumor rim. FIG. 11B shows a comparison ofthe Reference compound 1 (Gadovist®) at 0.3 mmol Gd/kg bw (triple dose)and Example 3 at 0.1 mmol Gd/kw bw (standard dose). Example 3 showedsimilar lesion-to-brain contrast at one third of the dose of Referencecompound 1.

1. A tetrameric gadolinium compound comprising: four1,4,7,10-tetraazacyclododecan-1-yl units complexed with fourgadolinium(III) ions, wherein an aqueous solution of the tetramericgadolinium compound has an r₁ relaxivity value of greater than 7.3 Lmmol⁻¹s⁻¹ and an r₂ relaxivity value of greater than 8.3 L mmol⁻¹s⁻¹ inwater at 1.41 T measured at 37° C.
 2. The tetrameric gadolinium compoundof claim 1, wherein the r₁ relaxivity value in water ranges from 9.4 to10.1 L mmol⁻¹s⁻¹ and the r₂ relaxivity value ranges from 10.8 to 11.7 Lmmol⁻¹s⁻¹ at 1.41 T measured at 37° C.
 3. The tetrameric gadoliniumcompound of claim 1, wherein the aqueous solution of the tetramericgadolinium compound has an r₁ relaxivity value of greater than 8.9 Lmmol⁻¹s⁻¹ in water at 3.0 T measured at 37° C.
 4. The tetramericgadolinium compound of claim 3, wherein the r₁ relaxivity value in waterranges from 8.9 to 9.2 L mmol⁻¹s⁻¹ at 3.0 T measured at 37° C.
 5. Thetetrameric gadolinium compound of claim 1, wherein the tetramericgadolinium compound has a structure selected from the group consistingof: Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,12,15-tetraoxo-16-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate;Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2S,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]propyl}amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate;Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate;and Tetragadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-{1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetrayltetrakis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}dodecaacetate,or a stereoisomer, a tautomer, a hydrate, a solvate, or a salt thereof,or a mixture of same.
 6. The tetrameric gadolinium compound of claim 5,wherein the tetrameric gadolinium compound has a structure selected fromthe group consisting of: Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; and Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2S,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate, or a stereoisomer, a tautomer, a hydrate, asolvate, or a salt thereof, or a mixture of same.
 7. A tetramericgadolinium compound comprising: four 1,4,7,10-tetraazacyclododecan-1-ylunits complexed with four gadolinium(III) ions, wherein an aqueoussolution of the tetrameric gadolinium compound has an r₁ relaxivityvalue of greater than 9.7 L mmol⁻¹s⁻¹ and an r₂ relaxivity value ofgreater than 11.3 L mmol⁻¹s⁻¹ in human plasma at 1.41 T measured at 37°C.
 8. The tetrameric gadolinium compound of claim 7, wherein the r₁relaxivity value in human plasma ranges from 10.4 to 11.8 L mmol⁻¹s⁻¹and the r₂ relaxivity value ranges from 13.1 to 14.7 L mmol⁻¹s⁻¹ at 1.41T measured at 37° C.
 9. The tetrameric gadolinium compound of claim 7,wherein the aqueous solution of the tetrameric gadolinium compound hasan r₁ relaxivity value of greater than 10.1 L mmol⁻¹s⁻¹ in human plasmaat 3.0 T measured at 37° C.
 10. The tetrameric gadolinium compound ofclaim 9, wherein the r₁ relaxivity value in human plasma ranges from10.1 to 11.4 L mmol⁻¹s⁻¹ at 3.0 T measured at 37° C.
 11. The tetramericgadolinium compound of claim 7, wherein the tetrameric gadoliniumcompound has a structure selected from the group consisting of:Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,12,15-tetraoxo-16-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({2-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate;Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2S,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[2-oxo-2-({3-({[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)-2,2-bis[({[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)methyl]propyl}amino)ethyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate;Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{2,5,11,14-tetraoxo-15-[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-8,8-bis({[({[4,7,10-tris-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}amino)acetyl]amino}methyl)-3,6,10,13-tetraazapentadec-1-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate;and Tetragadolinium2,2′,2″,2′″,2″″,2′″″,2″″″,2′″″″,2″″″″,2′″″″″,2″″″″″,2′″″″″″-{1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetrayltetrakis[(2-oxoethane-2,1-diyl)imino(1-oxopropane-1,2-diyl)-1,4,7,10-tetraazacyclododecane-10,1,4,7-tetrayl]}dodecaacetate,or a stereoisomer, a tautomer, a hydrate, a solvate, or a salt thereof,or a mixture of same.
 12. The tetrameric gadolinium compound of claim11, wherein the tetrameric gadolinium compound has a structure selectedfrom the group consisting of: Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; and Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2S,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate, or a stereoisomer, a tautomer, a hydrate, asolvate, or a salt thereof, or a mixture of same.
 13. An aqueouspharmaceutical solution comprising a tetrameric gadolinium compound:wherein the tetrameric gadolinium compound comprises four1,4,7,10-tetraazacyclododecan-1-yl units complexed with fourgadolinium(III) ions, wherein the aqueous pharmaceutical solutioncomprising a tetrameric gadolinium compound has an r₁ relaxivity valueof greater than 9.7 L mmol⁻¹s⁻¹ and an r₂ relaxivity value of greaterthan 11.3 L mmol⁻¹s⁻¹ in human plasma at 1.41 T measured at 37° C. 14.The aqueous pharmaceutical solution of claim 13, wherein the r₁relaxivity value in human plasma ranges from 10.4 to 11.8 L mmol⁻¹s⁻¹and the r₂ relaxivity value ranges from 13.1 to 14.7 L mmol⁻¹s⁻¹ at 1.41T measured at 37° C.
 15. The aqueous pharmaceutical solution of claim13, wherein the tetrameric gadolinium compound is Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate, or a stereoisomer, a tautomer, a hydrate, asolvate, or a salt thereof, or a mixture of same.
 16. The aqueouspharmaceutical solution of claim 13, wherein the tetrameric gadoliniumcompound has a structure selected from the group consisting of:Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium{4,10-bis(carboxylatomethyl)-7-[(2S,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl]-1,4,7,10-tetraazacyclododecan-1-yl}acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16S)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2R,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2S)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate; and Tetragadolinium[4,10-bis(carboxylatomethyl)-7-{(2S,16R)-3,6,12,15-tetraoxo-16-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-9,9-bis({[({(2R)-2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-yl]propanoyl}amino)acetyl]amino}methyl)-4,7,11,14-tetraazaheptadecan-2-yl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate, or a stereoisomer, a tautomer, a hydrate, asolvate, or a salt thereof, or a mixture of same.